Methods and compositions for treating coronavirus infections

ABSTRACT

The present application relates to a compositions and methods comprising or expressing a HEVAR or MOMO30 protein derived from Momordica balsamina. The HEVAR or MOMO30 protein and/or a nucleic acid encoding the same may be used in methods for treating or preventing viral infections, particularly those caused by coronaviruses, such as SARS-CoV-2.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/996,153, filed on Aug. 18, 2020. The entirety ofthe aforementioned applications is incorporated herein by reference.

FIELD

The present application generally relates to methods for treatingrespiratory infections. More particularly, the present applicationrelates to an antiviral composition comprising a plant HEVAR or MOMO30protein or nucleic acid product therefrom for treatment and preventionof coronavirus infections, such as SARS-CoV-2.

BACKGROUND

The surfaces of host cells and viruses are decorated by complex glycans,which play multifaceted roles in the dynamic interplay between the virusand the host including viral entry into host cell, modulation ofproteolytic cleavage of viral proteins, recognition and neutralizationof virus by host immune system (Raman, R. et al., Curr. Opin. Struct.Biol., 40: 153-162, 2016). These roles are mediated by specificmultivalent interactions between cell surfaces decorated by complexglycans and their cognate protein lectins.

Lectin proteins are sugar-binding proteins that bind specifically andreversibly to carbohydrate groups. They are typically anchored on thesurfaces of cells and are found in all groups of living organismsincluding plants, animals, fungi and bacteria, as well as viruses andmycoplasmas. Depending on their broad sugar-binding specificity, theyhave been classified as mannose-, galactose-, N-acetyl-glucosamine-,fucose- and sialic acid-binding lectins, according to the simple sugarsthat inhibit their carbohydrate-binding properties.

The complex glycans displayed on host cell surfaces typically act asattachment factors, co-receptors or primary receptors that arespecifically recognized by viral surface glycoprotein similarlydecorated by a variety of glycans. For example, complex glycansterminated by α2-3 or α2-6-linked sialic acid (N-acetyl neuraminic acid)act as receptors for several different viruses. Linear sulfatedglycosaminoglycans such as heparan sulfate act as co-receptors for avariety of viruses, including dengue virus, hepatitis C virus, andfoot-and-mouth disease virus. The display of specific glycan motifs onsurfaces of different cells and tissues contributes to the hostrestriction and cell/tissue tropism of viruses.

The complex glycans on the viral surface also play a key role in hostimmune response to counter the viral infection and play a dual role toenhance antigen presentation and processing for adaptive immuneresponses. In particular, sites of N-linked glycosylation are oftenpositively selected during evolution of a virus in human hosts toincrease glycans on the viral surface so as to present glycans thatmimic self-antigens and mask the underlying protein epitope which inturn permits the virus to evade host immune response.

A wide variety of lectins from animals, plants, algae, cyanobacteria andother sources have been shown to possess antiviral activity against awide variety of viruses, including coronaviruses, human immunodeficiencyviruses (HIVs), influenza viruses, herpes simplex viruses, Ebolaviruses, and others. See e.g., Mani et al., Virus Res., Apr. 30, 2020,pp. 197989; Akkouh et al., Molecules, 20:648-668, 2015). For example,mannose binding lectin (MBL), a serum protein in humans important inhost defenses has been shown to selectively bind to the SARS CoV Spike(S) protein in a SARS-CoV pseudotyped virus and potently inhibitSARS-CoV infection of susceptible cell lines at concentrations belowthose observed in the serum of healthy individuals (Zhou, Y et al., JVirol., 84(17): 8753-8764, 2010). Mutagenesis indicated that a singleN-linked glycosylation site, N330, was critical for the specificinteractions between MBL and SARS-S. Id. Exemplary lectins with broadspectrum antiviral activity against multiple viruses includeConcanavalin A from jack bean, Griffithsin from red algae, andCyanovirin-N from cyanobacteria.

The inventor of the present application has recently identified apotential broad spectrum antiviral agent termed MOMO30, which hasproperties characteristic of lectins. See co-pending U.S. patentapplication Ser. No. 16/718,994, filed Dec. 18, 2019, which is expresslyincorporated by reference herein. In particular, MOMO30 was found tobind HIV-1, simian immunodeficiency virus 1 (SIV-1), Ebola virus, andmurine leukemia virus (MuLV).

As of Jun. 3, 2020, the outbreak of SARS-CoV-2 infections, also known asCOVID-19, has affected 21,294,845 individuals, caused 761,779 deaths(WHO Situation report-209; Aug. 16, 2020), and has affected the entireworld (213 countries/areas/territories). The USA alone reported5,258,565 infected cases with the highest number of fatalities(n=167,201). Presently, there are virtually no FDA approved antiviralagents showing efficacy for treatment or prevention of coronavirusinfections, such as SARS-CoV-2. In view of the outbreak and its toll onhuman lives, there is a need for prophylactic and therapeutic optionsfor treating coronavirus infections, especially those caused bySARS-CoV-2.

SUMMARY

In one aspect, the present application relates to a method for treatingor preventing a viral infection, comprising orally administering to asubject in need thereof a pharmaceutical composition comprising aneffective amount of a hevamine A-related (HEVAR) protein which comprisesan amino acid sequence at least 90% identical to SEQ ID NO: 15 and atleast one pharmaceutically acceptable carrier. In one embodiment, theamino acid sequence is at least 95% identical to SEQ ID NO: 15. Inanother embodiment, the amino acid sequence comprises the amino acidsequence of SEQ ID NO: 15.

In another aspect, a method for preventing or reducing the severity ofthe cytokine storm in a SARS-CoV-2 infected patient comprisesadministering an effective amount of a pharmaceutical composition apharmaceutical composition comprising an effective amount of a (HEVARprotein which comprises an amino acid sequence at least 90% identical toSEQ ID NO: 15 and at least one pharmaceutically acceptable carrier,where the method results in a reduction of at least 10%, 20%, 50% or 80%in cytokine levels for one or more of IL-6, IL-1β, IL-2, IL-10, IFN-γ,TNF-α, GM-CSF, or VEGF. In one embodiment, the amino acid sequence is atleast 95% identical to SEQ ID NO: 15. In another embodiment, the aminoacid sequence comprises the amino acid sequence of SEQ ID NO: 15.

In preferred embodiments, the infection is caused by a Severe AcuteRespiratory Syndrome Corona Virus (SARS-CoV), such as SARS-CoV-2,SARS-CoV-1, or Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

In one embodiment, the composition is in a dried form, such as a capsuleor tablet. In another embodiment, the composition is in a liquid form.In a particular embodiment, the liquid form comprises an herbal tea.

In another embodiment, the composition comprises a second antiviralagent targeting a viral infection. In certain particular embodiments,the second antiviral agent targets an infection caused by SAR-CoV-2,SARS-CoV-1 or MERS.

In another aspect, a pharmaceutical composition comprises asubstantially pure HEVAR protein which comprises an amino acid sequenceat least 90% identical to SEQ ID NO: 15; and at least onepharmaceutically acceptable carrier. In one embodiment, the HEVARprotein comprises the amino acid sequence of SEQ ID NO: 15.

In one embodiment, the composition is in a dried form, such as a capsuleor tablet. In another embodiment, the composition is in a liquid form.In a particular embodiment, the liquid form comprises an herbal tea.

In one embodiment, the HEVAR protein is prepared by the steps of: (a)drying a plant comprising HEVAR protein; (b) extracting the dried plantin aqueous media; (c) lysing cells from the extracted plant to form aplant cell lysate; and (d) centrifuging the plant cell lysate to removedebris and particulates to form a clarified plant cell lysate; and (e1)passing the plant cell lysate through a molecular weight cut-off filterand collecting the HEVAR-containing retentate, or (e2) purifying theHEVAR protein from the clarified plant cell lysate by immunoaffinitypurification using an anti-HEVAR antibody.

In another aspect, the present application provides a compositioncomprising a nucleic acid having at least 95% or 100% identity to SEQ IDNO: 12 or SEQ ID NO: 13. In another embodiment. In a particularembodiment, the nucleic acid is operatively linked to an expressionvector.

In another aspect, the present application provides a cell comprising anucleic acid, plasmid or expression vector encoding an HEVAR protein.

In another aspect, the present application provides a method fortreating or preventing a viral infection, comprising orallyadministering to a subject in need thereof a pharmaceutical compositioncomprising: an effective amount of an expression vector operativelylinked to a nucleic acid having at least 95% or 100% identity to thenucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13; and at least onepharmaceutically acceptable carrier.

In another aspect, the present application relates to a method fortreating or preventing a coronavirus (CoV) infection, comprising orallyadministering to a subject in need thereof a composition comprising aneffective amount of a MOMO30 protein prepared by a method includes thesteps of: (a) drying plant comprising MOMO30 protein; (b) extracting thedried plant in aqueous media; (c) lysing cells from the extracted plantto form a plant cell lysate; (d) centrifuging the plant cell lysate toremove debris and particulates to form a clarified plant cell lysate;and (e) preparing the MOMO30 protein composition by: (i) passing theclarified plant cell lysate through a molecular weight cut-off filterand collecting the MOMO30-containing retentate; or (ii) purifying theMOMO30 protein from the clarified plant cell lysate by immunoaffinitypurification using an anti-MOMO30 antibody. The composition comprisingthe MOMO30 protein purified therefrom is orally administered to thesubject in liquid or dried form.

In certain preferred embodiments, the plant comprising MOMO30 is amember of the Momordica genus. In a more particular embodiment, theplant is Momordica balsamina.

In some embodiments, the method for preparing the MOMO30 proteincomposition comprises the step of subjecting the plant extract toimmunoaffinity purification prior to administration. In otherembodiments, the method includes the step of eluting the MOMO30retentate in an aqueous buffer to form an aqueous MOMO30 proteincomposition in solution.

In some embodiments, the MOMO30 protein composition is administered tothe subject in a dried form, such as a capsule or tablet. In otherembodiments, the MOMO30 protein composition is administered to thesubject in liquid form. In certain particular embodiments the MOMO30protein composition is formulated as an herbal tea for oraladministration in liquid form.

In another aspect, a method for preparing a MOMO30 protein composition,includes the steps of: (a) drying a plant comprising MOMO30 protein; (b)extracting the dried plant in aqueous media; (c) lysing cells from theextracted plant to form a plant cell lysate; (d) centrifuging the plantcell lysate to remove debris and particulates to form a clarified plantcell lysate; and (e1) passing the plant cell lysate through a molecularweight cut-off filter and collecting the MOMO30-containing retentate, or(e2) purifying the MOMO30 protein from the clarified plant cell lysateby immunoaffinity purification using an anti-MOMO30 antibody, such thatthe MOMO30 protein composition formed therefrom is substantially free ofplant components less than 10 kDa in size and includes a protein ofabout 30 kDa in size that is stable after boiling at 100° C. for 20 min,binds CoV S protein and is derived from a MOMO30 protein having a signalpeptide comprising the amino acid sequence of SEQ ID NO: 1.

In certain preferred embodiments, the plant comprising MOMO30 protein isa member of the Momordica genus. In a more particular embodiment, theplant is Momordica balsamina.

In another aspect, the present application provides a pharmaceuticalcomposition containing MOMO30 protein for treating or preventing acoronavirus infection in which the pharmaceutical composition isprepared by a method including the steps of: (a) drying a plantcomprising MOMO30 protein; (b) extracting the dried plant in aqueousmedia; (c) lysing cells from the extracted plant to form a plant celllysate; (d) centrifuging the plant cell lysate to remove debris andparticulates to form a clarified plant cell lysate; (e) preparing adried MOMO30 protein composition therefrom; (f) adding one or morepharmaceutically acceptable carriers to the dried MOMO30 proteincomposition, and (g) forming a pharmaceutically acceptable oralcomposition therefrom in the form of a powder, capsule, tablet, orliquid. The composition resulting therefrom is substantially free ofplant components less than 10 kDa in size, and includes a protein ofabout 30 kDa in size that is stable after boiling at 100° C. for 20 min,binds CoV S protein and is derived from a MOMO30 protein having signalpeptide comprising the amino acid sequence of SEQ ID NO: 1.

In certain preferred embodiments, the plant comprising MOMO30 protein isa member of the Momordica genus. In a more particular embodiment, theplant is Momordica balsamina.

In some embodiments, the clarified plant cell lysate is passed through a30-50 kDa molecular weight cut-off filter prior to preparing the driedMOMO30 protein composition. In other embodiments, the clarified plantcell lysate is subjected to immunoaffinity purification using ananti-MOMO30 antibody prior to preparing the dried MOMO30 proteincomposition for oral administration.

In some embodiments, the pharmaceutical composition is administered inthe form of a powder. In other embodiments, the pharmaceuticalcomposition is administered in the form of a capsule or tablet. In yetother embodiments, the pharmaceutical composition is administered in theform of a liquid.

In specific embodiments, the composition comprising the MOMO30 or HEVARprotein is about 30 kDa in size and is characterized by one orproperties such that the composition is substantially free of plantcomponents less than 10 kDa in size, is stable after boiling at 100° C.for 20 min, binds CoV S protein and comprises the HEVAR amino acidsequence of SEQ ID NO: 15 or is derived from a MOMO30 protein having asignal peptide sequence set forth in SEQ ID NO: 1.

In another aspect, a method for preventing or treating a viral infectioncomprises administering to a subject in need thereof, a MOMO30 protein,HEVAR protein, MOMO30-encoded nucleic acid or HEVAR-encoded nucleic acidby in vivo or ex vivo gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary process for producing an aqueous plant extractfrom dried Momordica balsamina leaves for purifying the MOMO30 or HEVARprotein.

FIG. 2A shows the N-terminal sequence of MOMO30 as determined by Edmandegradation. FIG. 2B shows the top ten hits when the N-terminal sequencewas compared to the NR database by BLAST (light blue). FIG. 2C is awestern blot showing detection of a 30 kDa protein from a M. balsaminaplant extract using a rabbit polyclonal antibody directed against theN-terminal amino acids of the MOMO30 protein in panel A.

FIG. 3 shows that MOMO30 causes hemagglutination. Purified MOMO30 wastested for its ability to agglutinate sheep red blood cells (RBCS). Thestock solution at a dilution of 1:512 was found to causehemagglutination.

FIG. 4 shows that MOMO30 stimulates T cell growth. In each experiment, afixed number of Jurkat cells was treated (left to right) with either PBS(control, Con), phytohemagglutinin A (PHA) or an equal amount of MOMO30.

FIG. 5 depicts the results from a MAGI cell indicator assay usingMOMO30-containing plant extract A in the presence of increasingconcentrations of the monosaccharide mannose where higher bars indicate“inactivation” of the inhibitory effect.

FIG. 6A shows a surface plasmon resonance (SPR) analysis (Biacore)indicating that MOMO30 protein from Extract A attaches to HIV gp120 soas to prevent its interaction with the CD4 receptor. Gp120 wasimmobilized on the gold surface and MOMO30 protein was flowed across thesurface at concentrations from 6 to 200 nM. The assay was done intriplicate on separate days.

FIG. 6B shows that binding of MOMO30 to gp120 is dependent on glycosylresidues on gp120. A Biocore chip was saturated with gp120 and MOMO30(top curves). The gp120-MOMO30 complexes were treated with PNGglycosylase to remove sugar residues from gp120 (bottom curves). Loss ofsugar residues resulted in a decrease in binding.

FIG. 7 shows the nucleotide sequence of the hevamine-related (HEVAR)coding region aligned with the hevamine A-related nucleotide sequencefrom Momordica charantia.

FIG. 8 panel A shows the amino acid sequence of the HEVAR coding regionaligned with the hevamine A-related amino acid sequence from Momordicacharantia. FIG. 8, panel B shows the amino acid sequence of the matureHEVAR (i.e. secreted) protein.

FIG. 9 shows an alignment of two conserved regions from the HEVARprotein against other hevamine A-related proteins.

While the present disclosure will now be described in detail, and it isdone so in connection with the illustrative embodiments, it is notlimited by the particular embodiments illustrated in the figures and theappended claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention and accompanying drawings will now be discussed inreference to the numerals provided therein to enable one skilled in theart to practice the present invention. The skilled artisan willunderstand, however, that the inventions described below can bepracticed without employing these specific details, or that they can beused for purposes other than those described herein. Indeed, they can bemodified and can be used in conjunction with products and techniquesknown to those of skill in the art considering the present disclosure.The drawings and descriptions are intended to be exemplary of variousaspects of the invention and are not intended to narrow the scope of theappended claims. Furthermore, it will be appreciated that the drawingsmay show aspects of the invention in isolation and the elements in onefigure may be used in conjunction with elements shown in other figures.

It will be appreciated that reference throughout this specification toaspects, features, advantages, or similar language does not imply thatall the aspects and advantages may be realized with the presentinvention should be or are in any single embodiment of the invention.Rather, language referring to the aspects and advantages is understoodto mean that a specific aspect, feature, advantage, or characteristicdescribed in connection with an embodiment is included in at least oneembodiment of the present invention. Thus, discussion of the aspects andadvantages, and similar language, throughout this specification may, butdo not necessarily, refer to the same embodiment.

The described aspects, features, advantages, and characteristics of theinvention may be combined in any suitable manner in one or more furtherembodiments. Furthermore, one skilled in the relevant art will recognizethat the invention may be practiced without one or more of the specificaspects or advantages of a particular embodiment. In other instances,additional aspects, features, and advantages may be recognized andclaimed in certain embodiments that may not be present in allembodiments of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs. One of skill in the art willrecognize many techniques and materials similar or equivalent to thosedescribed here, which could be used in the practice of the aspects andembodiments of the present application. The described aspects andembodiments of the application are not limited to the methods andmaterials described.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to “the value,” greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed.

As used herein, the term “preprotein” is used with reference to apredicted amino acid sequence including an N-terminal signal peptide,which is cleaved off during protein processing resulting in a secretedbiologically active mature protein as described herein.

As used herein, the term “MOMO30 protein” is used with reference to a 30kDa plant protein from Momordica balsamina that is stable after boilingor autoclaving at 120° C. for 20 min, and exhibits mannose-sensitivebinding to cell surface proteins. Additionally, a MOMO30 protein orMOMO30 homolog comprises an amino acid sequence that is 100%, 99.9%,99.5%, 99%, 95%, 94%, 93%, 92%, 91%, or 90 identical (including anypercent homology range therefrom) to the MOMO30 amino acid sequence ofSEQ ID NO: 1. Additional embodiments related to MOMO30 and applicable tothe present disclosure are described in co-pending U.S. patentapplication Ser. Nos. 16/718,994 and 16/996,153, the disclosures ofwhich are incorporated by reference in their entirety.

As used herein, the term “HEVAR protein” refers to a hevamine A-relatedprotein from Momordica balsamina. Additionally a “HEVAR protein” or“HEVAR homolog” comprises an amino acid sequence that is 100%, 99.9%,99.5%, 99%, 95%, 94%, 93%, 92%, 91%, or 90% identical (including anypercent homology range therefrom) to the HEVAR preprotein amino acidsequence of SEQ ID NO: 14 or the HEVAR mature amino acid sequence of SEQID NO: 15.

The phrase “cytokine storm” refers to an excessively activated cytokinecascade or hypercytokinemia, i.e., an excessive or uncontrolled releaseof proinflammatory cytokines, which can be associated with a widevariety of infectious and noninfectious diseases or disorders.

The phrase “antiviral agent” refers to a small molecule, protein orantibody that can inhibit the progression of coronavirus infections orinduce or mediate the death (e.g., necrosis or apoptosis) ofcoronavirus-infected cells in a subject (e.g., a human).

The terms “treat” and “treatment” refer to the amelioration of one ormore symptoms associated with a viral infection; prevention or delay ofthe onset of one or more symptoms of a viral infection; and/or lesseningof the severity or frequency of one or more symptoms of the infection.

The phrases “effective amount” “therapeutically effective”,“pharmacologically effective amount” are used interchangeably to meanthe amount(s) of one or more antiviral agents needed to provide athreshold level of active antagonist agents in the bloodstream or in thetarget tissue. The precise amount will depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, including based upon the information provided hereinor otherwise available in the relevant literature.

The phrases “pharmaceutical composition comprises” and “pharmaceuticalcomposition comprising” should be interpreted such that the “comprises”or “comprising” components are included in a single pharmaceuticalcomposition or in one or more independent pharmaceutical compositions.

The terms, “improve”, “increase” or “reduce”, as used in this context,indicate values or parameters relative to a baseline measurement, suchas a measurement in the same individual prior to initiation of thetreatment described herein, or a measurement in a control individual (ormultiple control individuals) in the absence of the treatment describedherein.

The term “control individual” is an individual who is not afflicted withthe same viral infection as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable). The individual (also referred to as “patient” or“subject”) being treated may be a fetus, infant, child, adolescent, oradult human.

Methods of Treatment

The present application provides a method for preventing or treatingviral infections, particularly those caused by respiratory viruses, suchas coronaviruses and influenza viruses.

In one aspect, the application relates to a method for treating orpreventing a viral infection, comprising orally administering to asubject in need thereof a pharmaceutical composition comprising: aneffective amount of hevamine A-related (HEVAR) protein which comprisesan amino acid sequence at least 90% identical to SEQ ID NO: 15; and atleast one pharmaceutically acceptable carrier. In one embodiment, theamino acid sequence is at least 95% identical to SEQ ID NO: 15. Inanother embodiment, the amino acid sequence comprises the amino acidsequence of SEQ ID NO: 15. The amino acid sequence of SEQ ID NO: 15corresponds to the mature (secreted) HEVAR protein from Momordicabalsamina. Exemplary symptoms include high fever, coughing, shortness ofbreath, inflammation (redness and swelling), hypercytokinemia,hypoxemia, low lung capacity and volume, lung fibrosis, fatigue, sorethroat, muscle pains, headache, runny nose, and nausea.

In a preferred embodiment, the virus is Severe Acute RespiratorySyndrome Coronavirus-2 (SARS-CoV-2). In other preferred embodiments, thevirus is Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1) orMiddle East Respiratory Syndrome Coronavirus (MERS-CoV).

In another aspect, a pharmaceutical composition comprises an HEVARprotein which comprises an amino acid sequence at least 90%, 95% or 99%identical to SEQ ID NO: 15; and at least one pharmaceutically acceptablecarrier. In one embodiment, the HEVAR protein comprises the amino acidsequence of SEQ ID NO: 15.

In one embodiment, the HEVAR-containing composition is in a dried form,such as a capsule or tablet. In another embodiment, the composition isin a liquid form. In a particular embodiment, the liquid form comprisesan herbal tea.

In one embodiment, the HEVAR protein is prepared by the steps of: (a)drying a plant comprising HEVAR protein; (b) extracting the dried plantin aqueous media; (c) lysing cells from the extracted plant to form aplant cell lysate; and (d) centrifuging the plant cell lysate to removedebris and particulates to form a clarified plant cell lysate; and (e1)passing the plant cell lysate through a molecular weight cut-off filterand collecting the HEVAR-containing retentate, or (e2) purifying theHEVAR protein from the clarified plant cell lysate by immunoaffinitypurification using an anti-HEVAR antibody.

In another aspect, the present application provides a compositioncomprising a nucleic acid having at least 90%, 95%, 99% or 100% identityto SEQ ID NO: 12.

In another aspect, the present application provides a cell comprising aplasmid or expression vector comprising a nucleic acid having at least90%, 95% or 100% identity to the nucleotide sequence of SEQ ID NO: 12.

In another aspect, the present application provides a method fortreating or preventing a viral infection, comprising orallyadministering to a subject in need thereof a pharmaceutical compositioncomprising: an effective amount of an expression vector operativelylinked to a nucleic acid having at least 95% identity to the nucleotidesequence of SEQ ID NO: 12; and at least one pharmaceutically acceptablecarrier.

In preferred embodiments, the virus is SARS-CoV-2, SARS-CoV-1 or MERS.

A major objective in treating coronavirus patients, particularlySARS-CoV-2 infected patients, is to reduce viral loads and prevent orreduce the severity of the cytokine storm and its potentially lethaleffects. A number of cytokines with anti-inflammatory properties areresponsible for the cytokine storm, such as IL-10 and transforminggrowth factor β (TGF-β). Each cytokine acts on a different part of theinflammatory response. For example, products of the Th2 immune responsesuppress the Th1 immune response and vice versa. By resolving theassociated inflammation, one can minimize collateral damage tosurrounding cells, with little or no long-term damage to the patient.

Accordingly, another aspect of the present application relates to amethod for preventing or reducing the severity of the cytokine storm ina SARS-CoV-2 infected patient comprises administering an effectiveamount of a pharmaceutical composition a pharmaceutical compositioncomprising an effective amount of a hevamine A-related (HEVAR) proteinwhich comprises an amino acid sequence at least 90% identical to SEQ IDNO: 15 and at least one pharmaceutically acceptable carrier, where themethod results in a reduction of at least 10%, 20%, 50% or 80% incytokine levels for one or more of IL-6, IL-113, IL-2, IL-10, IFN-γ,TNF-α, GM-CSF, or VEGF. In one embodiment, the amino acid sequence is atleast 95% identical to SEQ ID NO: 15. In another embodiment, the aminoacid sequence comprises SEQ ID NO: 15. To inhibit or reduce thecoronavirus-associated cytokine storm, the pharmaceutical compositionsof the present application may be administered alone or in combinationwith one or more antiviral agents, including those described below,including cytokine antagonists to the cytokines described herein.

In another aspect, a method for preventing or treating a viral infectioncomprises administering to a subject in need thereof a pharmaceuticalcomposition comprising MOMO30 protein, a MOMO30 extract,MOMO30-containing combination formulation or a MOMO30-encoded nucleicacid according to the present application to reduce the symptomsassociated with the infection or cure the subject of the disease. Inpreferred embodiments, the virus is SARS-CoV-2, SARS-CoV-1 or MERS-CoV.

The MOMO30 product is derived from Momordica balsamina and ischaracterized by multiple properties, including: (1) a size of about 30kDa; (2) solubility in aqueous solutions; (3) high heat resistance orhigh stability as reflected in no appreciable loss of activity followingautoclaving at 120° C. for 30 min; (4) exhibiting mannose sensitivebinding; (5) insensitive to digestion with trypsin followingdenaturation in 8M urea and overnight and partially sensitive tosubtilisin after overnight treatment; (6) having hemagglutinin activity;(7) capable of activating and stimulating T cell proliferation; (8)having chitinase activity; (9) having an amino terminal amino acidsequence of SEQ ID NO: 1, which is at least 93% identical to a hevamineA-related protein from Prosopis alba. In addition, the MOMO30 and HEVARproteins are believed to bind CoV S protein and exhibitmannose-sensitive binding to CoV S protein (data not shown).

In certain embodiments, the MOMO30 or HEVAR protein has an amino acidsequence that is 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQID NO: 1. In one embodiment, the MOMO30 protein is isolated from a plantof the Momordica genus, a species therefrom, such as Momordicabalsamina, or any homolog thereof.

Without wishing to be bound by theory, MOMO30 and HEVAR are believed tobe a carbohydrate binding agents with two distinct modes of action: (1)inhibition of virus by blocking entry into cells; (2) selecting formutations in a viral surface protein that allow the host to produce abroadly neutralizing antibody response. MOMO30 and HEVAR are believed toinhibit virus binding via its binding to carbohydrates, particularlycell surface mannose residues. The more carbohydrates on a surfaceprotein, such as CoV S protein (i.e., spike protein), the more targetswill be available for inhibiting virus. Under such pressure, thepresence of MOMO30 or HEVAR selects for viruses with fewer glycosylgroups. Fewer glycosyl groups on the CoV S protein allows more epitopesto be exposed and allows the production of neutralizing antibodies. As aconsequence, patients treated with HEVAR or MOMO30 in the short-termexhibit the production of a broadly neutralizing antibody response. Thesame patients should also develop a broadly neutralizing antibodyresponse to control their infection in the long term.

The compositions and methods of the present application may be appliedto any coronavirus in the Orthocoronavirinae family, including but notlimited to those described herein. The genetically diverseOrthocoronavirinae family is divided into four genera (alpha, beta,gamma, and delta coronaviruses). Human CoVs are limited to the alpha andbeta subgroups. Exemplary human CoVs include severe acute respiratorysyndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndromecoronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus(MERS-CoV), HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1.

Before the advent of human SARS CoV-2 (also known as COVID-19), humancoronaviruses were believed to cause 10% of all upper and lowerrespiratory tract infections, which typically present with common-coldlike symptoms, but were known to cause more severe disease in youngchildren, as well as people with underlying respiratory conditions (i.e.asthma, COPD) and the elderly.

Zoonotic CoVs have a natural predilection for emergence into new hostspecies giving rise to new diseases most recently exemplified in humansby severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severeacute respiratory syndrome coronavirus (SARS-CoV), and Middle Eastrespiratory syndrome coronavirus (MERS-CoV) (de Wit et al., 2016).Interestingly, all known human CoVs are thought to have emerged aszoonoses from wild or domestic animals.

Nonlimiting examples of subgroup 1a alphacoronaviruses and their GenBankAccession Nos. include FCov.FIPV.79.1146.VR.2202 (NV_007025),transmissible gastroenteritis virus (TGEV) (NC_002306; Q811789.2;DQ811786.2; DQ811788.1; DQ811785.1; X52157.1; AJ011482.1; KC962433.1;AJ271965.2; JQ693060.1; KC609371.1; JQ693060.1; JQ693059.1; JQ693058.1;JQ693057.1; JQ693052.1; JQ693051.1; JQ693050.1); porcine reproductiveand respiratory syndrome virus (PRRSV) (NC 001961.1; DQ811787), as wellas any subtype, clade or sub-clade thereof, including any other subgroup1a coronavirus now known (e.g., as can be found in the GenBank®Database) or later identified in the GenBank® Database.

Nonlimiting examples of a subgroup 1b alphacoronaviruses and theirGenBank Accession Nos. include HCoV.NL63.Amsterdam.I (NC_005831),BtCoV.HKU2.HK.298.2006 (EF203066), BtCoV.HKU2.HK.33.2006 (EF203067),BtCoV.HKU2.HK.46.2006 (EF203065), BtCoV.HKU2.GD.430.2006 (EF203064),BtCoV.1A.AFCD62 (NC_010437), BtCoV.1B.AFCD307 (NC_010436),BtCov.HKU8.AFCD77 (NC_010438), BtCoV.512.2005 (DQ648858); porcineepidemic diarrhea viruses (NC_003436, DQ355224.1, DQ355223.1,DQ355221.1, JN601062.1, JN601061.1, JN601060.1, JN601059.1, JN601058.1,JN601057.1, JN601056.1, JN601055.1, JN601054.1, JN601053.1, JN601052.1,JN400902.1, JN547395.1, FJ687473.1, FJ687472.1, FJ687471.1, FJ687470.1,FJ687469.1, FJ687468.1, FJ687467.1, FJ687466.1, FJ687465.1, FJ687464.1,FJ687463.1, FJ687462.1, FJ687461.1, FJ687460.1, FJ687459.1, FJ687458.1,FJ687457.1, FJ687456.1, FJ687455.1, FJ687454.1, FJ687453.1, FJ687452.1,FJ687451.1, FJ687450.1, FJ687449.1, AF500215.1, KF476061.1, KF476060.1,KF476059.1, KF476058.1, KF476057.1, KF476056.1, KF476055.1, KF476054.1,KF476053.1, KF476052.1, KF476051.1, KF476050.1, KF476049.1, KF476048.1,KF177258.1, KF177257.1, KF177256.1, KF177255.1), HCoV.229E (NC_002645),as well as any subtype, clade or sub-clade thereof, including any othersubgroup 1b coronavirus now known (e.g., as can be found in the GenBank®Database) or later identified in the GenBank® Database.

Nonlimiting examples of subgroup 2a betacoronaviruses and their GenBankAccession Nos. include HCoV.HKU1.C.N5 (DQ339101), MHV.A59 (NC_001846),PHEV.VW572 (NC_007732), HCoV.OC43.ATCC.VR.759 (NC_005147), bovineenteric coronavirus (BCoV.ENT) (NC_003045), as well as any subtype,clade or sub-clade thereof, including any other subgroup 2a coronavirusnow known (e.g., as can be found in the GenBank® Database) or lateridentified in the GenBank® Database.

Nonlimiting examples of subgroup 2b betacoronaviruses and their GenBankAccession Nos. include human SARS CoV-2 isolates, such as Wuhan-Hu-1(NC_045512.2) and any CoV-2 isolates comprising a genomic sequence setforth in GenBank Accession Nos., such as MT079851.1, MT470137.1,MT121215.1, MT438728.1, MT470115.1, MT358641.1, MT449678.1, MT438742.1,LC529905.1, MT438756.1, MT438751.1, MT460090.1, MT449643.1, MT385425.1,MT019529.1, MT449638.1, MT374105.1, MT449644.1, MT385421.1, MT365031.1,MT385424.1, MT334529.1, MT466071.1, MT461669.1, MT449639.1, MT415321.1,MT385430.1, MT135041.1, MT470179.1, MT470167.1, MT470143.1, MT365029.1,MT114413.1, MT192772.1, MT135043.1, MT049951.1; human SARS CoV-1isolates, such as SARS CoV.A022 (AY686863), SARSCoV.CUHK-W1 (AY278554),SARSCoV.GD01 (AY278489), SARSCoV.HC.SZ.61.03 (AY515512), SARSCoV.SZ16(AY304488), SARSCoV.Urbani (AY278741), SARSCoV.civet010 (AY572035),SARSCoV.MA.15 (DQ497008); bat SARS CoV isolates, such as BtSARS.HKU3.1(DQ022305), BtSARS.HKU3.2 (DQ084199), BtSARS.HKU3.3 (DQ084200),BtSARS.Rm1 (DQ412043), BtCoV.279.2005 (DQ648857), BtSARS.Rf1 (DQ412042),BtCoV.273.2005 (DQ648856), BtSARS.Rp3 (DQ071615),), as well as anysubtype, clade or sub-clade thereof, including any other subgroup 2bcoronavirus now known (e.g., as can be found in the GenBank® Database)or later identified in the GenBank® Database.

Nonlimiting examples of subgroup 2c betacoronaviruses and their GenBankAccession Nos. include Middle East respiratory syndrome coronavirus(MERS) isolates, such as Riyadh 22012 (KF600652.1), Al-Hasa_18_2013(KF600651.1), Al-Hasa_17_2013 (KF600647.1), Al-Hasa_15_2013(KF600645.1), Al-Hasa_16_2013 (KF600644.1), Al-Hasa_21_2013 (KF600634),Al-Hasa_19_2013 (KF600632), Buraidah_1_2013 (KF600630.1),Hafr-Al-Batin_1_2013 (KF600628.1), Al-Hasa_12_2013 (KF600627.1),Bisha.ltoreq.1_2012 (KF600620.1), Riyadh_3_2013 (KF600613.1),Riyadh_1_2012 (KF600612.1), Al-Hasa_3_2013 (KF186565.1), Al-Hasa_1_2013(KF186567.1), Al-Hasa_2_2013 (KF186566.1), Al-Hasa_4_2013 (KF186564.1);Betacoronavirus England 1-N1 (NC_019843), SA-N1 (KC667074); humanbetacoronavirus 2c Jordan-N3/2012 (KC776174.1); human betacoronavirus 2cEMC/2012, (JX869059.2); any bat coronavirus subgroup 2c isolate, such asbat coronavirus Taper/CII_KSA_287/Bisha/Saudi Arabia (KF493885.1), batcoronavirus Rhhar/CII_KSA 003/Bisha/Saudi Arabia/2013 (KF493888.1), batcoronavirus Pikuh/CII_KSA_001/Riyadh/Saudi Arabia/2013 (KF493887.1), batcoronavirus Rhhar/CII_KSA 002/Bisha/Saudi Arabia/2013 (KF493886.1), batcoronavirus Rhhar/CII_KSA_004/Bisha/Saudi Arabia/2013 (KF493884.1), batcoronavirus BtCoV.HKU4.2 (EF065506), bat coronavirus BtCoV.HKU4.1(NC_009019), bat coronavirus BtCoV.HKU4.3 (EF065507), bat coronavirusBtCoV.HKU4.4 (EF065508), bat coronavirus BtCoV133.2005 (NC_008315), batcoronavirus BtCoV.HKU5.5 (EF065512), bat coronavirus BtCoV.HKU5.1(NC_009020), bat coronavirus BtCoV.HKU5.2 (EF065510), bat coronavirusBtCoV.HKU5.3 (EF065511), and bat coronavirus HKU5 isolate (KC522089.1);any additional subgroup 2c, such as KF192507.1, KF600656.1, KF600655.1,KF600654.1, KF600649.1, KF600648.1, KF600646.1, KF600643.1, KF600642.1,KF600640.1, KF600639.1, KF600638.1, KF600637.1, KF600636.1, KF600635.1,KF600631.1, KF600626.1, KF600625.1, KF600624.1, KF600623.1, KF600622.1,KF600621.1, KF600619.1, KF600618.1, KF600616.1, KF600615.1, KF600614.1,KF600641.1, KF600633.1, KF600629.1, KF600617.1, KC869678.2, KC522088.1,KC522087.1, KC522086.1, KC522085.1, KC522084.1, KC522083.1, KC522082.1,KC522081.1, KC522080.1, KC522079.1, KC522078.1, KC522077.1, KC522076.1,KC522075.1, KC522104.1, KC522104.1, KC522103.1, KC522102.1, KC522101.1,KC522100.1, KC522099.1, KC522098.1, KC522097.1, KC522096.1, KC522095.1,KC522094.1, KC522093.1, KC522092.1, KC522091.1, KC522090.1, KC522119.1,KC522118.1, KC522117.1, KC522116.1, KC522115.1, KC522114.1, KC522113.1,KC522112.1, KC522111.1, KC522110.1, KC522109.1, KC522108.1, KC522107.1,KC522106.1, KC522105.1); Pipistrellus bat coronavirus HKU4 isolates(KC522048.1, KC522047.1, KC522046.1, KC522045.1, KC522044.1, KC522043.1,KC522042.1, KC522041.1, KC522040.1, KC522039.1, KC522038.1, KC522037.1,KC522036.1, KC522048.1, KC522047.1, KC522046.1, KC522045.1, KC522044.1,KC522043.1, KC522042.1, KC522041.1, KC522040.1, KC522039.1, KC522038.1,KC522037.1, KC522036.1, KC522061.1, KC522060.1, KC522059.1, KC522058.1,KC522057.1, KC522056.1, KC522055.1, KC522054.1, KC522053.1, KC522052.1,KC522051.1, KC522050.1, KC522049.1, KC522074.1, KC522073.1, KC522072.1,KC522071.1, KC522070.1, KC522069.1, KC522068.1, KC522067.1, KC522066.1,KC522065.1, KC522064.1, KC522063.1, KC522062.1), as well as any subtype,clade or sub-clade thereof, including any other subgroup 2c coronavirusnow known (e.g., as can be found in the GenBank® Database) or lateridentified in the GenBank® Database.

Nonlimiting examples of subgroup 2d betacoronaviruses and their GenBankAccession Nos. include BtCoVIIKU9.2 (EF065514), BtCoV.HKU9.1(NC_009021), BtCoV.HkU9.3 (EF065515), BtCoV.HKU9.4 (EF065516), as wellas any subtype, clade or sub-clade thereof, including any other subgroup2d coronavirus now known (e.g., as can be found in the GenBank®Database) or later identified in the GenBank® Database.

Nonlimiting examples of subgroup 3 gammacoronaviruses includeIBV.Beaudette.IBV.p65 (DQ001339) or any other subgroup 3 coronavirus nowknown (e.g., as can be found in the GenBank® Database) or lateridentified in the GenBank® Database.

A coronavirus defined by any of the isolates or genomic sequences in theaforementioned subgroups 1a, 1b, 2a, 2b, 2c, 2d and 3 can be targetedfor prophylactic or therapeutic use in accordance with the methods andcompositions of the present application.

The methods of the present application may be also be used to prevent ortreat other viral infections that are inhibited by the HEVAR protein,MOMO30 protein, HEVAR-encoded expression vector or a MOMO30-encodedexpression vector. Viruses for treatment include enveloped RNA and DNAviruses. In certain preferred embodiments, the virus includes one ormore surface proteins containing mannose residues.

In addition to coronaviruses, exemplary RNA viruses for prophylactic ortherapeutic treatment include retroviruses (e.g., HIV-1, HIV-2, HTLV-I,HTLV-II); bunyaviruses (e.g., Rift Valley fever virus, Crimean-Congohemorrhagic fever virus); filoviruses (e.g., Ebola virus, Marburgvirus); flaviviruses (e.g., Hepatitis C virus, West Nile virus, Denguefever virus, Zika virus, yellow fever virus, tick-borne encephalitisvirus, Saint Louis encephalitis virus, GB virus C); enteroviruses (TypesA to L, including coxsackieviruses (Types A to C), echoviruses,rhinoviruses (Types A to C), poliovirus); orthomyxoviruses (e.g.,influenza Types A, -B, -C, -D, including A subtypes H1N1, H5N1, H3N2);paramyxoviruses (e.g., rubulavirus (mumps), rubeola virus (measles),respiratory syncytial virus, Newcastle disease, parainfluenza);parvoviruses (e.g., parvovirus B19 virus); rhabdoviruses (e.g., Rabiesvirus); arenaviruses (e.g., lymphocytic choriomeningitis virus andseveral Lassa fever viruses, including Guanarito virus, Junin virus,Lassa virus, Lujo virus, Machupo virus, Sabia virus, Whitewater Arroyovirus); alphaviruses (e.g., Venezuelan equine encephalitis virus,eastern equine encephalitis virus; western equine encephalitis virus);Hepatitis A virus; Hepatitis D virus; Hepatitis E virus; as well as anytype, subtype, clade or sub-clade thereof.

In other preferred embodiments, the RNA virus for prevention ortreatment is a respiratory virus, such as influenza Type A virus.Influenza A viruses are divided into subtypes on the basis of twoproteins on the surface of the virus, hemagglutinin (HA) andneuraminidase (NA). There are 18 known HA subtypes and 11 known NAsubtypes. Many different combinations of HA and NA proteins arepossible. For example, an “H7N2 virus” designates an influenza A virussubtype that has an HA7 protein and an NA2 protein. Similarly, an “H5N1”virus has an HA5 protein and an NA1 protein. Type A influenza virusesthat may be targeted for prophylactic and/or therapeutic use accordingto the methods and compositions of the present application include avariety of sub-types, such as H1N1, H1N2, H3N2, H5N1, H5N2, H5N3, H5N4,H5N5, H5N6, H5N7, H5N8, and H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6,H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9,H17N10 and H18N11).

In one embodiment, a method for treating or preventing an influenza TypeA virus infection, comprises orally administering to a subject in needthereof a composition comprising: an effective amount of a HEVAR proteinwhich comprises an amino acid sequence that is at least 90% identical toSEQ ID NO: 15; and at least one pharmaceutically acceptable carrier.

Exemplary DNA viruses for prophylactic or therapeutic treatment includeherpesviruses (e.g., HSV-1, HSV-2, EBV, VZV, HCMV-1, HHV-6, HHV-7,HHV-8), papillomaviruses (e.g., human papilloma virus (HPV) Types 1, 2,4, 6, 11, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 41, 42, 43, 44, 45,51, 52, 54, 55, 56, 57, 58, 59, 61, 62, 64, 67, 68, 69, 70); poxviruses(e.g., smallpox virus), hepadnaviruses (Hepatitis B virus);anelloviruses (e.g., transfusion transmitted virus or torque teno virus(TTV)); as well as any type, subtype, clade or sub-clade thereof.

Route and Dose of Antiviral Product Administration

The antiviral MOMO30 or HEVAR product of the present application may beadministered orally, intrathecally, intra-arterially, intravenously,intradermally, subcutaneously, transdermally (topically) ortransmucosally. An antiviral composition may be administered by anyroute, including oral, rectal, pulmonary, sublingual, and parenteraladministration. Parenteral administration includes, for example,intraperitoneal, intravenous, intramuscular, intraarterial, intravesical(e.g., to the bladder), intradermal, transdermal, topical, orsubcutaneous administration.

As a general proposition, the therapeutically effective amount of anantiviral MOMO30 or HEVAR protein administered will be in a weight rangeof about 1 ng/kg body weight/day to about 100 mg/kg body weight/daywhether by one or more administrations. In more particular embodiments,the antiviral MOMO30 protein or HEVAR protein is administered in weightrange from about 1 ng/kg body weight/day to about 1 μg/kg bodyweight/day, 1 ng/kg body weight/day to about 100 ng/kg body weight/day,1 ng/kg body weight/day to about 10 ng/kg body weight/day, 10 ng/kg bodyweight/day to about 1 μg/kg body weight/day, 10 ng/kg body weight/day toabout 100 ng/kg body weight/day, 100 ng/kg body weight/day to about 1μg/kg body weight/day, 100 ng/kg body weight/day to about 10 μg/kg bodyweight/day, 1 μg/kg body weight/day to about 10 μg/kg body weight/day, 1μg/kg body weight/day to about 100 μg/kg body weight/day, 10 μg/kg bodyweight/day to about 100 μg/kg body weight/day, 10 μg/kg body weight/dayto about 1 mg/kg body weight/day, 100 μg/kg body weight/day to about 10mg/kg body weight/day, 1 mg/kg body weight/day to about 100 mg/kg bodyweight/day and 10 mg/kg body weight/day to about 100 mg/kg bodyweight/day.

In other embodiments, an antiviral MOMO30 or HEVAR protein isadministered at a dosage range of 1 ng-10 ng per injection, 10 ng-100 ngper injection, 100 ng-1 μg per injection, 1 μg-10 μg per injection, 10μg-100 μg per injection, 100 μg-1 mg per injection, 1 mg-10 mg perinjection, 10 mg-100 mg per injection, and 100 mg-1000 mg per injection.The MOMO30 protein, HEVAR protein, or formulation thereof, may beinjected once daily, twice daily, three times daily, and/or every 2, 3,4, 5, 6 or 7 days. In addition, the MOMO30 or HEVAR protein orformulation thereof may be administered over a period of one month, twomonths, six months, 12 months, 2 years, 5 years, 10 years, 20 years, ormore.

In other embodiments, the antiviral MOMO30 protein, HEVAR protein, orformulation thereof may be administered in a range from about 1 ng/kg toabout 100 mg/kg. In more particular embodiments, the antiviral MOMO30protein, HEVAR protein, or formulation thereof may be administered in arange from about 1 ng/kg to about 10 ng/kg, about 10 ng/kg to about 100ng/kg, about 100 ng/kg to about 1 μg/kg, about 1 μg/kg to about 10ng/kg, about 10 μg/kg to about 100 μg/kg, about 100 μg/kg to about 1mg/kg, about 1 mg/kg to about 10 mg/kg, about 10 mg/kg to about 100mg/kg, about 0.5 mg/kg to about 30 mg/kg, and about 1 mg/kg to about 15mg/kg.

In other particular embodiments, the amount of the antiviral MOMO30 orHEVAR protein is administered is, or is about, 0.0006, 0.001, 0.003,0.006, 0.01, 0.03, 0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300,600 and 1000 mg/day.

Concentrations or amounts of MOMO30 or HEVAR protein may be determinedusing anti-MOMO30 or anti-HEVAR antibodies as further described hereinbelow. The specific dose of antiviral MOMO30 or HEVAR product may bedetermined based on the particular circumstances of the individualpatient including the size, weight, age and sex of the patient, thenature and stage of the disease, the aggressiveness of the disease, andthe route of administration of the antiviral composition.

In certain embodiments, an antiviral MOMO30 product, HEVAR product,MOMO30-containing formulation or HEVAR-containing formulation may beadministered at least once per day, typically once, twice, three timesor four times per day with the doses given at equal intervals throughoutthe day and night in order to maintain a constant presence of the drugin order to provide sufficient antiviral activity. However, a skilledartisan will appreciate that a treatment schedule can be optimized forany given patient, and that administration of compound may occur lessfrequently than once per day.

In other embodiments, an antiviral MOMO30 product, HEVAR product,MOMO30-containing formulation or HEVAR-containing formulation of thepresent application may be administered in combination with otherantiviral agents. Exemplary antiviral agents for treating coronavirusinfections include viral polymerase inhibitors, such as Remdesivir,GS-441524, Faviravir, EIDD-2801, EIDD-2901, EIDD-1931, Ribavirin,6-azauridine; convalescent plasma; neutralizing mAbs or mAbs inhibitingcoronavirus attachment or entry, such as REGN10933, REGN10987,LY3819253, AZD7442, BRII-196, CT-P59, JS016, SCTA01, STI-1499, TY027,47D11; anti-IL6 monoclonal antibodies, such as Tocilizumab; proteaseinhibitors targeting M^(pro), such as Lopinavir, Ritonavir,Dipyridamole, and Danoprevir; Ivermectin; Saracatinib; proteaseinhibitors targeting TMPRSS2, such as Camostat, Nafomastat, andNafomastat mesylate; S-protein targeted drugs, such as Arbidol(umifenovir) and Hydroxychloroquine; cytokines, such as interferons-α,-β, -λ, Dexamethasone, Anakinra, Hydrocortisone, Azithromycin,Ulinastatin and Ciclesonide; Janus kinase inhibitors, such asRuxolitinib and Baricitinib; AXL kinases inhibitors, such asBemcentinib; Dihydroorotate dehydrogenase (DHODH) inhibitors, such asPTC299; recombinant ACE2; HR2P-EK1C4, IPB03 and other lipopeptide fusioninhibitors described herein above; Fluvoxamine; Apilomod; Ciclesonide;Tetrahydroquinoline oxocarbazate; GC373; Vitamin D; Zn²⁺; andcombinations thereof. When used in such combinations, each of theantiviral MOMO30 or HEVAR product may be co-administered with theantiviral agent(s) simultaneously, separately, by the same or differentroutes, and/or at different times during treatment.

The treatment may be carried out for as long a period as necessary,i.e., until the infection is cleared or no longer a threat to the host.In some cases, the treatment may be continued indefinitely while thedisease state persists, although discontinuation might be indicated ifthe antiviral compositions no longer produce a beneficial effect. In oneembodiment, the treatment is carried out for 6 months and thendiscontinued. The treating physician can determine whether to increase,decrease, or interrupt treatment based on a patient's response,including evaluation of immune responses, viral loads etc.

MOMO30 and HEVAR Products for Use in the Present Application

In another aspect, the present application contemplates plant homologsor variants of MOMO30 or HEVAR having about 80% to about 100% nucleotideor amino acid sequence identity to a complete MOMO30 or HEVAR nucleotideor amino acid sequence, including any and all whole numbers within, aswell as any subranges within, wherein the lower number can be any wholenumber between 81% and 99% and the upper number can be any whole numberbetween 82% and 100%.

In some embodiments, the MOMO30 or HEVAR protein (or homolog thereof) isencoded by a plant species of the Momordica genus. Exemplary Momordicaspecies include, but are not limited to, M. aculeata, M. acuminate, M.acutangula, M. adoensis, M. affinis, M. amaniana, M. angolensis, M.angulate, M. angustisepala, M. anigosantha, M. anthelmintica, M.argillicola, M. aspera, M. auriculata, M. balsamina, M. bequaertii, M.bicolor, M. boivinii, M. brachybotrys, M. bracteata, M. brevispinosa, M.bricchettii, M. cabraei, M. calantha, M. calcarata, M. camerounensis, M.cardiospermoides, M. carinata, M. casea, M. charantia, M. chinensis, M.cirrhiflora, M. cissoides, M. clarkeana, M. clematidea, M.cochinchinensis, M. cochinchinensis, M. cogniauxiana, M. cordata, M.cordatifolia, M. coriacea, M. corymbifera, M. covel, M. crinocarpa, M.cucullata, M. cylindrica, M. cymbalaria, M. dasycarpa, M. denticulata, Mdenudata, M. dictyosperma, M. dioica, M. diplotrimera, M. dissecta, M.eberhardtii, M. echinata, M. echinocarpa, M. ecirrhata, M. elastica, M.elaterium, M. elegans, M. enneaphylla, M erinocarpa, M. fasciculata, M.foetida, M. friesiorum, M. gabonii, M. garipensis, M. garriepensis, M.gilgiana, M. glabra, M. glauca, M. gracilis, M. grandibracteata, M.grosvenorii, M. guttata, M. hamiltoniana, M. hamiltoniana, M.henriquesii, M. heterophylla, M. heyneana, M. hispida, M. huberi, M.humilis, M. hystrix, M. indica, M. involucrata, M. jagorana, Mjeffreyana, M. kirkii, M. lambertiana, M. lanata, M. laotica, M.laurentii, M. leiocarpa, M. littorea, M. luffa, M. luffa, M. macrantha,M. macropetala, M. macrophylla, M. macropoda, M. macrosperma, M.maculata, M. mannii, M. marlothii, M. martinicensis, M. melonifiora, M.microphylla, M. missionis, M. mixta, M. monadelpha, M. morkorra, M.mossambica, M. multicrenulata, M. multiflora, M. muricata, M.obtusisepala, M. officinarum, M. operculata, M. ovata, M. paina, M.palmata E, M. papillosa, M. parvifolia, M. pauciflora, M. pedata, M.pedisecta, M. peteri, M. procera, M. pterocarpa, M. punctata, M.purgans, M. pycnantha, M. quinquefida, M. quinqueloba, M. racemiflora,M. racemosa, M. renigera, M. repens, M. reticulata, M. rostrata, M.rotunda, M. roxburghiana, M. rumphii, M. runssorica, M. rutshuruensis,M. sahyadrica, M. sativa, M. schimperiana, M. schinzii, M. schliebenii,M. senegalensis, M. sessilifolia, M. sicyoides, M. silvatica, M.sinensis, M. somalensis, M. sphaeroidea, M. spicata, M. spinosa, M.stefaninii, M. subangulata, M. surculata, M. suringarii, M thollonii, M.tonkinensis, M. trifolia, M. trifoliata, M. trilobata, M. tuberosa, M.tubiflora, M. tubulosa, M. umbellata, M. verticillata, M. vogelii, M.wallichii, M. welwitschii, M. wildemaniana, M. zeylanica, and M.zeylanica. In some embodiments, the MOMO30 or HEVAR protein may beobtained from any of the foregoing Momordica leaf extracts, fruitextracts, root extracts, bark extracts, seed extracts and/or any flowerthereof.

In certain preferred embodiments, the MOMO30 or HEVAR protein isobtained from Momordica balsamina leaf extracts. In other embodiments,the MOMO30 or HEVAR protein is obtained from Momordica balsamina fruitextracts, root extracts, bark extracts, seed extracts and/or any flowerthereof. In yet other embodiments, the MOMO30 or HEVAR protein isprepared from cells transformed with an expression vector encoding M.balsamina MOMO30 or HEVAR, or any other MOMO30 or HEVAR plant source.

In other embodiments, the MOMO30 or HEVAR protein (or homolog thereof)is encoded by a plant species of the Prosopis genus. Exemplary Prosopisspecies include, but are not limited to, P. abbreviata, P. affinis, P.african, P. alba, P. chilensis, P. cineraria, P. farcta, P. fiebrigii,P. flexuosa, P. glandulosa, P. hassleri, P. julifiora, P. laevigata, P.koelziana, P. kuntzei, P. nigra, P. pallida, P. pubescens, P. reptans,P. rojasiana, P. ruscifolia, P. spicigera, P. strombulifera, P.tamarugo, and P. velutina. In some embodiments, the MOMO30 protein maybe obtained from any of the foregoing Prosopis leaf extracts, fruitextracts, root extracts, bark extracts, seed extracts and/or any flowerthereof.

In some embodiments, the MOMO30 or HEVAR protein is 100%, 99.9%, 99.5%,99%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,82%, 81%, or 80% identical to the amino acid sequence of the Momordicabalsamina MOMO30 or HEVAR protein, including any range therefrom.

In some embodiments, the MOMO 30 or HEVAR protein is a variantcontaining one or more mutations relative to the wild-type sequence.“Variants” include protein sequences having one or more amino acidadditions, deletions, stop positions, or substitutions, as compared to awild-type protein. An amino acid substitution can be a conservative or anon-conservative substitution. Variants of MOMO30 or HEVAR proteins caninclude those having one or more conservative amino acid substitutions.A “conservative substitution” or “conservative amino acid substitution”involves a substitution found in one of the following conservativesubstitutions groups: Group 1: Alanine (Ala; A), Glycine (Gly; G),Serine (Ser; S), Threonine (Thr; T); Group 2: Aspartic acid (Asp; D),Glutamic acid (Glu; E); Group 3: Asparagine (Asn; N), Glutamine (Gln;Q); Group 4: Arginine (Arg; R), Lysine (Lys; K), Histidine (His; H);Group 5: Isoleucine (Ile; I), Leucine (Leu; L), Methionine (Met; M),Valine (Val; V); and Group 6: Phenylalanine (Phe; F), Tyrosine (Tyr; Y),Tryptophan (Trp; W).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function, chemical structure, or composition (e.g.,acidic, basic, aliphatic, aromatic, or sulfur-containing). For example,an aliphatic grouping may include, for purposes of substitution, G, A,V, L, and I. Other groups including amino acids that are consideredconservative substitutions for one another include: sulfur-containing: Mand C; acidic: D, E, N, and Q; small aliphatic, nonpolar or slightlypolar residues: A, S, T, P, and G; polar, negatively charged residuesand their amides: D, N, E, and Q; polar, positively charged residues: H,R, and K; large aliphatic, nonpolar residues: M, L, I, V, and C; andlarge aromatic residues: F, Y, and W.

Non-conservative substitutions include those that significantly affect:the structure of the peptide backbone in the area of the alteration(e.g., the alpha-helical or beta-sheet structure); the charge orhydrophobicity of the molecule at the target site; or the bulk of theside chain. Non-conservative substitutions which in general are expectedto produce the greatest changes in a protein's properties may includethose in which e.g., (i) a hydrophilic residue (e.g., S or T) issubstituted for (or by) a hydrophobic residue (e.g. L, I, F, V, or A);(ii) a C or P is substituted for (or by) any other residue; (iii) aresidue having an electropositive side chain (e.g. K, R, or H) issubstituted for (or by) an electronegative residue (e.g., Q or D); or(iv) a residue having a bulky side chain (e.g., F) that is substitutedfor (or by) one not having a bulky side chain, (e.g., G).

MOMO30 and HEVAR mutants may be generated by random mutagenesis orsite-directed mutagenesis using methods known to those of ordinary skillin the art with or without selection methodologies employing bindingassays, functional assays, apoptosis assays and the like.

In some embodiments, the present application provides a nucleic acidencoding a MOMO30 or HEVAR protein of the present application. In someembodiments, the MOMO30- or HEVAR-encoded nucleic acid is 99.9%, 99%,95%, 94%, 93%, 92%, 91% or 90% identical to a Momordica balsamina MOMO30or HEVAR cDNA. In certain particular embodiments, the MOMO30- orHEVAR-encoded nucleic acid includes a codon-optimized MOMO30 or HEVARnucleic acid encoding the 30 kDa M. balsamina MOMO30 or HEVAR protein.

In some embodiments, the present application provides an expressionvector comprising a MOMO30- or HEVAR-encoded nucleic acid.

In other embodiments, the present application provides a host celltransformed with a MOMO30- or HEVAR-encoded nucleic acid or a MOMO30- orHEVAR-encoded expression vector.

The HEVAR protein, MOMO30 protein, HEVAR-encoded nucleic acid orMOMO30-encoded nucleic acid may be administered with a pharmaceuticallyacceptable carrier, alone or in combination with a suitable adjuvant, orit may be administered as a plant extract alone or in combination withother nutritional supplements, plant extracts, plant components orsecondary antiviral agents.

In another aspect, the present application provides an extractcomprising a MOMO30 or HEVAR protein. The extract may prepared for oraladministration or parenteral administration (e.g., intravenous (IV),intramuscular (IM), subcutaneous (SC or SQ), transdermal (TD)). Theextract may be in dried form or it may be in aqueous solution, with orwithout one or more pharmaceutically acceptable carriers. In oneembodiment, the extract is an herbal extract from a natural plantsource, such as M. balsamina. In another embodiment, the extract is acell extract from bacterial, fungal, plant, insect, or animal cellstransformed with a MOMO30 or HEVAR expression vector to express theprotein. The transformed cells may be stably transformed or they may betransiently transformed. The extract may be prepared and its compositionmay be modified in accordance with any of the methods of preparationoutlined below or known to those of ordinary skill in the art.

In some embodiments, the MOMO30 protein, HEVAR protein,MOMO30-containing extract or HEVAR-containing extract is combined withone or more nutritional supplements selected from the group consistingof minerals and metals, vitamins, salts, amino acids, fatty acids,proteins, and other pharmaceutically acceptable excipients. Thenutritional supplement may be included with the MOMO30 protein, HEVARprotein, MOMO30-containing extract or HEVAR-containing extract as in aMOMO30 or HEVAR formulation, or it may be separately administeredtherewith. Exemplary supplements include vitamin A, vitamin B1, vitaminB2, vitamin B5, vitamin B6, vitamin B12, vitamin C, magnesium citrate,vitamin E, vitamin D3, calcium, zinc citrate, selenium, manganesegluconate, copper gluconate, copper gluconate, Coenzyme Q, biotin,folate, acetyl-L-carnitine, chromium polynicotinate, citrusbioflavinoids, glucosamine sulfate, boron sulfate, and whey protein.Exemplary fatty acids may be selected from the group consisting oflinoleic acid (LA), gamma linoleic acid (GLA), eicosapentaneoic acid(EPA), docosapentaneoic Acid (DPA), docosahexaenoic acid (DHA), andD-alpha-tocopherol.

Alternatively, or in addition, in some embodiments, the MOMO30 protein,HEVAR protein, MOMO30-containing extract or HEVAR-containing extract maybe combined with one or more MOMO30 or HEVAR homologs, plant extractsand/or or plant substances to form a MOMO30 or HEVAR combinationformulation. Exemplary plant extracts, MOMO30 homologs and/or HEVARhomologs in such combination formulations may be obtained from one ormore members selected from the group consisting of Acacia arabia,Afromomum melegueta, Agrimonia eupatoria, Ajuga decumbens, Allium cepa,Allium sativum, Aloe vera, Alternanthera philoxeroides or sessiles, Ammimaius, Andographis paniculata, Apium graveolens, Apium leptophyllum,Arachis hypogaea, Arctium lappa, Artemesia Judaica, Amebia euhcroma,Asparagus racemosus, Astragalus spinosus, Astragalus lentingosisswainsonine, Azadirachta indica, Balanites aegyptiaca, Bauhiniarufescens, Bersama tysoniana, Blumea alata, Brucea antidysenterica,Buchenavia capita, Butyrospermum parkii, Bryonia cretica ssp. Dioica,Bryonia angustifolia, Calotropis procera, Camellia theifera, Camelliasinensis, Casia sieberiana, Catha edulis. Cedrela toona, Chrysanthemummorifolium, Clausena anisata, Clivia miniata, Cochlospermum planchonii,Coffea arabica, Cola nitida, Combretum glutinosum, Combretum micranthum,Coptis chinesis, Coptis teetoides, Coptis japonica, Coraria nepalensis,Coriandrum sativum, Cryptolepis sanguinolenta, Curcuma longa, Cyperusarticulatus, Cyperus domestus, Cyperus rigidifolius, Datura motel synalba, Daucus carota, Diospyros mespiliformis, Echinacea angustiflora andpurpurea, Echinacea simulata, Echinacea pallida, Entada abyssinica,Epimedium grandiflorum, Epimedium sagittatum, Epimedium sinense,Epilobium angustifolium, Erigeron Canadensis, Eugenia or Syzigiumclaviflorum, Euphorbia hirta, Faidherbia albida, Fagara xanthox, Ficusiteophylla, Ficus platphylla, Foeniculum vulgarel, Garcinia afzelii,Garcinia epundata, Gardenia coronaria, Gaultheria trichophylla, Glycinemax, Glycyrrhiza labra, Gossypium herbaceum, Guiera senegalensis,Heracleum sphondylium, Hypericum perforatum, Hypericum japonicum,Hyssopus officinalis, Jasminum officinale, Khaya senegalensis, Lippiajavanica, Lithospermum erythrorhizon, Lonicera japonica, Lophiralanceolate, Luffa, Lycopus europaeus, Magnolia officinalis, Mallotusrepandus, Mallotus philippinesis, Matricaria chamomil, Matricariarecutitia, Melissa parviflora, Melissa officinalis, Momordica species,including Momordica balsamina, Momordica charantia and others; Morindalucida, Narcissus tazetta, Narcissus pseudonarcissus, Ocimumgratissimum, Oenthera rosea, Paeonia spec., Papaver somniferum, Parkiabiglobosa, Perilla frutescens, Persea Americana, Phyllanthus niruri,Pinus koraicenis, Pinus parvifiora, Piper nirgum, Plumeria rubra,Polyantha suberosa, Prosopis sp., including P. africana and others;Prunus africans, Prunella vulgaris, Prunus bakariensis, Prunusamygdalus, Psoralea corylifolia, Randia dunatorum, Raphanus sativus,Rheum palmatum, Rhus coriaria, Rhus chinesis, Ricinus communis,Rosmarinus officinalis, Salic mucronata, Salvia miltiorhiza andofficinalis, Salvadora persica, Sambucus ebulus, Saussurea lappa, Scalagriffithii, Scutellaria baicalensis baiealein, Sedum sediforme, Senecioscandens, Senecio aereus, Senna alata, Silybum marianum, Skimmialaureola, Solarium niporum, Swertia franchetiana, Tamarindus indica,Terminalia alata, Terminalia catappa, Terminalia chebula, Terminaliaglaucescens, Thula occidentalis, Trapalaponica spec., Trichosanthesdioica, Trichosanthes kirilowii, Urtica dioica, Viola yeodensis,Vitellaria paradoxa, Voacanga africana, Woodfordia fruticosa, Woodwardiaspec., Zanoxylum nitidum, Zanthoxylum zanthoxyloides, and Ziziphusmauritania, including powder or extract from leaf, bark, seed, root,and/or flower therefrom.

In one embodiment, the MOMO30 or HEVAR combination formulation includesone or more plant extracts selected from the group consisting ofMomordica balsamina, Aframomum melegueta, Cyperus domestus, Ficusiteophylla and Tamarindus indica. In another embodiment, the MOMO30 orHEVAR combination formulation includes one or more plant extractsselected from the group consisting of Momordica balsamina, Aframomummelegueta, Cyperus articulatus, Ficus iteophylla and Tamarindus indica.In another embodiment, the MOMO30 or HEVAR combination formulationincludes one more plant extracts selected from the group consisting ofMomordica balsamina, Aframomum melegueta and Cyperus articulatus. In amore particular embodiment, the MOMO30 or HEVAR combination formulationincludes a leaf extract from Momordica balsamina, a seed extract fromAframomum melegueta and/or a root extract from Cyperus articulatus.

Exemplary plant-derived substances include lentinan, a polysaccharideisolated from the fruit body of shiitake mushroom (Lentinula edodesmycelium) and various ribosome inactivating proteins (RIPS) from M.balsamina and Trichosanthis kirilowii, such as Momordin I and MomordinII, as well as ribosome inactivating proteins from any of the foregoingplant extracts. It is believed that the addition of the aforementionednutritional supplements and/or plant-based substances may be furtherincrease the prophylactic and/or therapeutic efficacy of the MOMO30 orHEVAR protein, especially in patients infected with a CoV or any of theother viral infections described herein below.

Another aspect of the application is a method of preparing a MOMO30- orHEVAR-containing plant extract, including but not limited to plants ofthe Mormordica genus, such as Momordica balsamina. In one embodiment,the method includes one or more steps including: harvesting the plants;drying the plants; extracting the dried plants in water or aqueousmedia; collecting the plant cells by centrifugation; lysing ordisrupting the plant cell membranes by physical or chemical means;centrifuging the plant cell lysate to remove debris and particulates;filtering the clarified plant cell lysates by e.g., running the lysatethrough a molecular weight cutoff (MWCO) filter (e.g., Amicon 30 kDa or50 kDa); eluting the semi-purified extract from the retentate; dryingthe semi-purified extract or resuspending the semi-purified extract inbuffer for further analysis, purification and/or storage. The MOMO30protein may be further purified from the plant extract by immunoaffinitychromatography and other conventional methodologies known to those ofskill in the art.

In a particular embodiment, a method for preparing a partially purifiedMOMO30- or HEVAR-containing plant extract comprises the steps of: (a)forming an aqueous plant extract from one or more dried plant leavescomprising a MOMO30 and/or HEVAR protein; (b) lysing the plant cells;(c) centrifuging the aqueous plant extract to remove debris andparticulates; (d) retaining the aqueous supernatant; (e) filtering theaqueous supernatant through a MW cutoff filter and/or subjecting thesupernatant to immunoaffinity purification; and (f) eluting MOMO30 orHEVAR into buffer for storage and/or use. In certain embodiments, theMOMO30 or HEVAR protein may be dried for storage or resuspended in anappropriate buffer for further use or storage following e.g.,quantification of MOMO30 or HEVAR yield and/or characterization ofMOMO30 or HEVAR purity. In practice, the extracts are quite stable andhave been stored freeze dried for years without significant loss ofanti-viral activity.

MOMO30- or HEVAR containing cell extracts, as well as purified MOMO30 orHEVAR proteins may be characterized by HPLC and/or tested for bindingand/or functional activities via binding assays, infectivity assays andthe like. In some embodiments, the MOMO30- or HEVAR-containing plantextract or purified MOMO30 or HEVAR protein preparation may be evaluatedfor coronavirus binding activity using commercially availablecoronavirus reagents, cell lines and/or inhibitor screening assay kits.As further described below, the reagents and kits for these assays mayutilize a variety of SARS-CoV-2 S protein-, SARS-CoV-2 S1 subunit(receptor binding domain, RBD) protein-, and/or ACE2 protein reagents,which may be His-tagged, Fc-tagged, Avi-tagged, or biotin-labeled inorder to facilitate detection of binding on microtiter plates and thelike using suitable colorimetric, chemoluminescent substrates (BPSBioscience, San Diego, Calif.).

In one embodiment, MOMO30- or HEVAR containing plant extract or purifiedMOMO30 or HEVAR protein is evaluated for functional activity in an invitro plaque reduction assay using SARS-CoV-2 infected cells as furtherdescribed below.

In another embodiment, a MOMO30- or HEVAR containing plant extract orpurified MOMO30 or HEVAR protein is evaluated for its ability to inhibitinfection by a lentivirus operably linked to a luciferase reporter thatis pseudotyped with a CoV Spike (S) protein, such as SARS-CoV-2 Sprotein, in ACE2-expressing cells. A “bald” or non-pseudotypedlentivirus control containing the luciferase reporter alone can be usedas a negative control. These lentivirus vectors, as well as a lentivirusexpressing ACE2 can be obtained from BPS Bioscience, San Diego, Calif.,BPS #s 79942, 79943 and 79944).

In certain preferred embodiments, the plant leaves comprising MOMO30 orHEVAR protein are obtained from members of the Momordica genus. In amore particular embodiment, the plant leaves are obtained from theMomordica balsamina plant.

An antiviral MOMO30 or HEVAR protein of the present application can bechemically synthesized or produced from cells transiently or stablytransformed with polynucleotide expression vectors operatively linked toa MOMO30 or HEVAR gene using recombinant DNA technologies well known tothose skilled in the art. Polynucleotide expression vectors can bedesigned to facilitate preparative expression levels in many differentcell hosts, including bacteria, yeast, insect cells, and mammaliancells.

In some microorganisms, such as wild type E. coli, the periplasmconstitutes an oxidizing environment, whereas the cytoplasm is areducing environment. Accordingly, expression in the E. coli periplasmmay enable the production of peptides containing interchain orintrachain disulfide bonds that might be otherwise reduced in cytoplasm,where it may be toxic to the cell. Some prokaryotic organisms haveendogenous, intracellular oxidizing environments and can normallyaccommodate formation of protein disulfide bonds inside the cell.Accordingly, the fusion protein may be periplasmically expressed usingan operably linked periplasmic signal sequence at the 5′ end of thecorresponding nucleic acid expression construct.

The MOMO30 or HEVAR protein or nucleic acid therefrom may be fused toother protein domains, including binding tags conferring additionalbiochemical properties, targeting properties, antiviral properties etc.When fused to another protein domain in an expression vector, the MOMO30or HEVAR encoded nucleic acid may be further engineered to include acleavage recognition site for proteolytic cleavage of one or morepeptide domains from one another. The cleavage recognition sequence canbe cleaved by a suitable protease, such as Kex2p or furin, at one ormore defined residues.

Where the cleavage recognition site is positioned adjacent to a proteindomain, proteolytic cleavage in a transduced cell can liberate one ormore antiviral domains from one another so that the antiviral productscan function independently of one another according to their designatedmicrobial cell surface target or microbial intracellular target.

For example, when positioned in or adjacent to a spacer region adjacentto the MOMO30 or HEVAR gene product, the expressed protein can bedirectly cleaved when introduced into a microbial cell bearing thecorresponding protease. In one embodiment, the proteolytic recognitionsite is a Kex2p-sensitive proteolytic cleavage site. In anotherembodiment, the proteolytic recognition site is the furin proteolyticcleavage site, which is sensitive to cleavage by the enzyme, furin.

An expression construct can further include a native or non-nativeN-terminal signal peptide region to facilitate entry of the encodedantiviral MOMO30 or HEVAR protein into the secretory pathway followinggene transfer into eukaryotic cells near a site of infection.

Expression Vectors

In certain embodiments, an expression vector encoding the antiviralMOMO30 or HEVAR protein of the present application is directlyadministered to a patient to express an antiviral MOMO30 or HEVARprotein in vivo. In certain particular embodiments, a recombinantpolynucleotide operatively linked to suitable regulatory elements forexpression of a MOMO30 or HEVAR protein is codon optimized forexpression in a selected prokaryotic or eukaryotic host cell, such as amammalian, plant or insect cell. To facilitate replication andexpression, the polynucleotide can be incorporated into a vector, suchas a prokaryotic or a eukaryotic expression vector. Suitable non-viralexpression vectors include, but are not limited to, plasmid expressionvector or a bacteriophage vectors. Suitable viral vectors include, butare not limited to, adeno-associated viral (AAV) vectors, retroviralvectors, lentiviral vectors, adenoviral vectors, herpes viral vectors,and alphavirus vectors. The viral vector can also be an astrovirus,coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus,picornavirus, poxvirus, togavirus viral vector.

The term “in vivo expression vector” refers to a non-viral or viralvector that comprises a polynucleotide encoding an antiviral MOMO30 orHEVAR protein of the present application in a form suitable forexpression of the polynucleotide in a host cell. The expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, and operably linked to thepolynucleotide sequence to be expressed. It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors can be introduced into host cells to produce an antiviral MOMO30or HEVAR protein of the present application.

As used herein, the term “control sequences” or “regulatory sequences”refers to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism. The term“control/regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Control/regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Anexpression vector may be designed to facilitate expression of anantiviral MOMO30 or HEVAR protein-encoding polynucleotide in one or morecell types. Tissue-specific regulatory elements may be used to restrictexpression to a particular cell type.

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, a DNA for a presequence or secretory leaderpeptide is operably linked to DNA for a protein if it is expressed as apreprotein that participates in the secretion of the protein; a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous and, in the case of a secretory leader,contiguous and in reading phase. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers are used in accordance with conventional practice.

Delivery of antiviral MOMO30 or HEVAR protein-encoding expressionvectors can be achieved by infection (for viral vectors), transfection(for non-viral vectors) and other methods well known to one skilled inthe art. Examples of other delivery methods and media include,polycationic condensed DNA linked or unlinked to killed viruses, ligandlinked DNA, liposomes, eukaryotic cell delivery vehicles cells,deposition of photopolymerized hydrogel materials, handheld genetransfer particle gun, ionizing radiation, nucleic charge neutralizationor fusion with cell membranes. Particle mediated gene transfer may alsobe employed.

Plasmid DNA expression vectors can be utilized for non-viral genetransfer, either by direct injection of naked DNA or by encapsulating anantiviral MOMO30 or HEVAR protein-encoding polynucleotide in a liposome,microparticle, microcapsule, virus-like particle, or erythrocyte ghost.Such compositions can be further linked by chemical conjugation to, forexample, microbial translocation domains and/or targeting domains tofacilitate targeted delivery and/or entry of nucleic acids into thenucleus of desired cells to promote gene expression. In addition,plasmid vectors may be incubated with synthetic gene transfer moleculessuch as polymeric DNA-binding cations like polylysine, protamine, andalbumin, and linked to cell targeting ligands such as asialoorosomucoid,insulin, galactose, lactose or transferrin. Naked DNA may also beemployed. Uptake efficiency of naked DNA may be improved usingbiodegradable latex beads. Such delivery may be improved further bytreating the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.

As used herein, the term “promoter” is to be taken in its broadestcontext and includes transcriptional regulatory elements (TREs) fromgenomic genes or chimeric TREs therefrom, including the TATA box orinitiator element for accurate transcription initiation, with or withoutadditional TREs (i.e., upstream activating sequences, transcriptionfactor binding sites, enhancers and silencers) which regulate activationor repression of genes operably linked thereto in response todevelopmental and/or external stimuli and trans-acting regulatoryproteins or nucleic acids. The promoter may be constitutively active orit may be active in one or more tissues or cell types in adevelopmentally regulated manner. A promoter may contain a genomicfragment or it may contain a chimera of one or more TREs combinedtogether.

Examples of such promoters include: the immediate early promoter of CMV,LTR or SV40 promoter, polyhedron promoter of baculovirus, E. coli lac ortrp promoter, phage T7 and lambda PL promoter and other promoters knownto control expression of genes in prokaryotic or eukaryotic cells ortheir viruses. The expression vector typically also contains a ribosomebinding site for translation initiation and a transcription terminator.The vector optionally includes appropriate sequences for amplifyingexpression. In addition, the expression vectors optionally comprise oneor more selectable marker genes to provide a phenotypic trait forselection of transformed host cells, such as dihydrofolate reductase orneomycin resistance for eukaryotic cell culture or such as tetracyclineor ampicillin resistance in E. coli.

The expression vector can also include additional expression elements,for example, to improve the efficiency of translation. These signals caninclude, e.g., an ATG initiation codon and adjacent sequences. In somecases, for example, a translation initiation codon and associatedsequence elements are inserted into the appropriate expression vectorsimultaneously with the polynucleotide sequence of interest (e.g., anative start codon). In such cases, additional translational controlsignals are not required. However, in cases where only a protein codingsequence or a portion thereof, is inserted, exogenous translationalcontrol signals, including an ATG initiation codon is provided forexpression of an antiviral MOMO30 or HEVAR protein. The initiation codonis placed in the correct reading frame to ensure translation of thepolynucleotide sequence of interest. Exogenous transcriptional elementsand initiation codons can be of various origins, both natural andsynthetic. If desired, the efficiency of expression can be furtherincreased by the inclusion of enhancers appropriate to the cell systemin use (Scharf et al. (1994) Results Probl Cell Differ 20:125-62; Bitteret al. (1987) Methods in Enzymol 153:516-544).

Expression vectors carrying an antiviral MOMO30- or HEVAR-encodingnucleic acid can be introduced into host cells by any of a variety ofwell-known procedures, such as electroporation, liposome mediatedtransfection, calcium phosphate precipitation, infection, transfectionand the like, depending on the selection of vectors and host cells.

Host cells that contain antiviral MOMO30 or HEVAR protein-encodingnucleic acids are, thus, also a feature of this disclosure. Favorablehost cells include prokaryotic (i.e., bacterial) host cells, such as E.coli, as well as numerous eukaryotic host cells, including plant (e.g.,tobacco), fungal (e.g., yeast, such as Saccharomyces cerevisiae andPicchia pastoris) cells, insect cells, and mammalian cells (such as CHOcells). Recombinant antiviral MOMO30- or HEVAR-encoding nucleic acidsare introduced (e.g., transduced, transformed or transfected) into hostcells, for example, via a vector, such as an expression vector. Asdescribed above, the vector is most typically a plasmid, but suchvectors can also be, for example, a viral particle, a phage, etc.Examples of appropriate expression hosts include: bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium; fungal cells, suchas Saccharomyces cerevisiae, Pichia pastoris and Neurospora crassa;insect cells such as Drosophila and Spodoptera frugiperda; mammaliancells such as 3T3, COS, CHO, BHK, HEK 293 or Bowes melanoma; plantcells, including algae cells, etc.

The host cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants oramplifying the inserted polynucleotide sequences. The cultureconditions, such as temperature, pH and the like, are typically thosepreviously used with the host cell selected for expression and will beapparent to those skilled in the art.

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the expressed product. For example,when large quantities of a protein or fragments thereof are needed forthe production of antibodies, vectors which direct high level expressionof fusion proteins that are readily purified are favorably employed.Such vectors include, but are not limited to, multifunctional E. colicloning and expression vectors such as BLUESCRIPT (Stratagene), in whichthe coding sequence of interest, e.g., a polynucleotide of the inventionas described above, can be ligated into the vector in-frame with e.g.,sequences for the amino-terminal translation initiating methionine andthe subsequent 7 residues of beta-galactosidase producing acatalytically active beta galactosidase fusion protein in which theamino-terminal methionine is ligated in frame with a histidine tag; andthe like.

Similarly, in yeast, such as Saccharomyces cerevisiae, a number ofvectors containing constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH can be used for production of thedesired expression products. In mammalian host cells, a number ofexpression systems, including both plasmids and viral-based systems, canbe utilized.

A host cell is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, glycosylation, acetylation, carboxylation,phosphorylation, lipidation, acylation etc. Post-translationalprocessing for example, which cleaves a precursor form into a matureform of the protein (for example, by a furin protease) is optionallyperformed in the context of the host cell. Different host cells such as3T3, COS, CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and can be chosen to ensure the correct modification andprocessing of the introduced, foreign protein.

For long-term, high-yield production of recombinant antiviral MOMO30 orHEVAR protein, stable expression systems may be employed. For example,polynucleotides encoding an antiviral MOMO30 or HEVAR protein can beintroduced into suitable host cells using expression vectors whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells are allowed to grow for 1-2 days in an enriched media before theyare switched to selective media. The purpose of the selectable marker isto confer resistance to selection and its presence allows growth andrecovery of cells which successfully express the introduced sequences.For example, resistant groups or colonies of stably transformed cellscan be proliferated using tissue culture techniques appropriate to thecell type. Host cells transformed with a nucleic acid encoding anantiviral MOMO30 or HEVAR protein are optionally cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture.

Following transduction of a suitable host cell line and growth of thehost cells to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. The secretedprotein product is then recovered from the culture medium.Alternatively, cells can be harvested by centrifugation, disrupted byphysical or chemical means and the resulting crude extract retained forfurther purification. Eukaryotic or microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption or useof cell lysing agents or other methods, which are well known to thoseskilled in the art.

Expressed antiviral MOMO30 or HEVAR proteins can be recovered andpurified from recombinant cell cultures by any of a number of methodswell known in the art, including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography (e.g., using any of the tagging systems notedherein), hydroxylapatite chromatography and lectin chromatography. TheMOMO30 or HEVAR protein is unusually heat stable; this suggests thatapplication of heat to denature such proteins or homologs thereof may bea useful approach to protein purification. Protein refolding steps canbe used, as desired, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedin the final purification steps.

In certain examples, the nucleic acids are introduced into vectorssuitable for introduction and expression in prokaryotic cells, e.g., E.coli cells. For example, a nucleic acid including a polynucleotidesequence that encodes a F2GF1 chimeric RSV antigen can be introducedinto any of a variety of commercially available or proprietary vectors,such as the pET series of expression vectors (e.g., pET19b and pET21d).Expression of the coding sequence is inducible by IPTG, resulting inhigh levels of protein expression. The polynucleotide sequence encodingthe chimeric RSV antigen is transcribed under the phage T7 promoter.Alternate vectors, such as pURV22 that include a heat-inducible lambdapL promoter are also suitable.

The expression vector is introduced (e.g., by electroporation) into asuitable bacterial host. Numerous suitable strains of E. coli areavailable and can be selected by one of skill in the art (for example,the Rosetta and BL21 (DE3) strains have proven favorable for expressionof recombinant vectors containing polynucleotide sequences that encodeF2GF1 chimeric RSV antigens.

In another example, a polynucleotide sequence that encodes an antiviralproduct is introduced into insect cells using a baculovirus expressionvector system (BEVS). Recombinant baculovirus capable of infectinginsect cells can be generated using commercially available vectors, kitsand/or systems, such as the BD BaculoGold system from BD BioScience.Briefly, the polynucleotide sequence encoding the antiviral product isinserted into the pAcSG2 transfer vector. Then, host cells SF9(Spodoptera frugiperda) are co-transfected by pAcSG2-chimer plasmid andBD BaculoGold, containing the linearized genomic DNA of the baculovirusAutographa californica nuclear polyhedrosis virus (AcNPV). Followingtransfection, homologous recombination occurs between the pACSG2 plasmidand the Baculovirus genome to generate the recombinant virus. In oneexample, the antiviral product is expressed under the regulatory controlof the polyhedrin promoter (pH). Similar transfer vectors can beproduced using other promoters, such as the basic (Ba) and p10promoters. Similarly, alternative insect cells can be employed, such asSF21 which is closely related to the SF9 and the High Five (Hi5) cellline derived from a cabbage looper, Trichoplusia ni.

Following transfection and induction of expression (according to theselected promoter and/or enhancers or other regulatory elements), theexpressed proteins are recovered (e.g., purified or enriched) andrenatured to ensure folding into a biologically active conformation.

In yet other embodiments, the antiviral products are expressed in vivousing viral or non-viral expression vectors. In some embodiments, theantiviral hevamine-related proteins are delivered from viral-derivedexpression vectors. Exemplary viral vectors may include or be derivedfrom adeno-associated virus, adenovirus, herpesvirus, vaccinia virus,poliovirus, poxvirus, HIV virus, lentivirus, retrovirus, Sindbis andother RNA viruses and the like. Also preferred are any viral familieswhich share the properties of these viruses which make them suitable foruse as vectors. Retroviruses include Murine Moloney Leukemia virus(MMLV), HIV and other lentivirus vectors. Adenovirus vectors arerelatively stable and easy to work with, have high titers and can bedelivered in aerosol formulation and can transfect non-dividing cells.Poxviral vectors are large and have several sites for inserting genes,they are thermostable and can be stored at room temperature. Viraldelivery systems typically utilize viral vectors having one or moregenes removed and with and an exogenous gene and/or gene/promotercassette being inserted into the viral genome in place of the removedviral DNA. The necessary functions of the removed gene(s) may besupplied by cell lines which have been engineered to express the geneproducts of the early genes in trans.

In other embodiments, nonviral delivery systems are utilized fordelivery of plasmid vectors or other bioactive non nucleic acid agentsusing lipid formulations comprising, for example, liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) and anionicliposomes. Liposomes can be further conjugated to one or more proteinsor peptides to facilitate targeting to a particular cell, if desired.Administration of a composition comprising a compound and a cationicliposome can be administered to the blood afferent to a target organ orinhaled into the respiratory tract to target cells of the respiratorytract. Furthermore, active agent(s) can be administered as a componentof a microcapsule or nanoparticle that can be targeted to a cell type ofinterest using targeting moieties described herein or that can bedesigned for slow release of one or more active agent(s) in accordancewith a predetermined rate of release or dosage.

In other embodiments, the nucleic acids may be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.), as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

The nucleic acids may be in solution or suspension (for example,incorporated into microparticles, liposomes or cells). These may betargeted to a particular cell type via antibodies, receptors or receptorligands. Vehicles such as “stealth” and other antibody conjugatedliposomes (including lipid mediated drug targeting to cells ofinterest), receptor mediated targeting of DNA through cell specificligands or viral vectors targeting e.g., lymphoid, epithelial orendothelial cells. In general, receptors are involved in pathways ofendocytosis, either constitutive or ligand induced. These receptorscluster in clathrin-coated pits, enter the cell via clathrin-coatedvesicles, pass through an acidified endosome in which the receptors aresorted and then either recycle to the cell surface, become storedintracellularly or are degraded in lysosomes. The internalizationpathways serve a variety of functions, such as nutrient uptake, removalof activated proteins, clearance of macromolecules, opportunistic entryof viruses and toxins, dissociation and degradation of ligand andreceptor level regulation. Many receptors follow more than oneintracellular pathway, depending on the cell type, receptorconcentration, type of ligand, ligand valency and ligand concentration.

Pharmaceutical Compositions

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, fillers,stabilizers, binders, absorbents, bases, buffering agents, lubricants,controlled release, vehicles, diluents, emulsifying agents, humectants,lubricants, dispersion media, coatings, antibacterial or antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well-known in the art.See e.g., A. H. Kibbe Handbook of Pharmaceutical Excipients, 3rd ed.Pharmaceutical Press, London, UK (2000). Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary agentscan also be incorporated into the compositions. In certain embodiments,the pharmaceutically acceptable carrier comprises serum albumin.

The pharmaceutical composition of the application is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intrathecal, intra-arterial,intravenous, intradermal, subcutaneous, oral, transdermal (topical) andtransmucosal administration.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine; propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose, pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene,glycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an antiviral peptide) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the hevamine-related products aredelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forsustained or controlled release of the active ingredients.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and poly lactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from e.g., Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Suitable unit dosage forms include, but are notlimited to powders, tablets, pills, capsules, lozenges, suppositories,patches, nasal sprays, injectables, implantable sustained-releaseformulations, lipid complexes, etc.

A “dosage unit form” refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present application are dictated by and directly dependent on theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals.

Toxicity and therapeutic efficacy of the antiviral product of thepresent application can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50. Compounds whichexhibit large therapeutic indices are preferred. While compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the present application, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

The present application is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures and Tables, are incorporatedherein by reference.

EXAMPLES

Preparation of MOMO30-Containing Cell Extracts and Proteins

FIG. 1 shows an exemplary method for preparing a plant extract frome.g., dried M. balsamina plants for purifying MOMO30 or HEVAR protein.In one non-limiting embodiment, the method comprises the steps of: (a)forming an aqueous plant extract from a dried M. balsamina plant; (b)lysing plant cells therefrom so as to form an aqueous plant extract; (c)centrifuging the aqueous plant extract to remove debris and particulatesand retaining the aqueous supernatant; (d) filtering the aqueoussupernatant through a MW cutoff filter to sterilize and further purifythe MOMO30 or HEVAR protein; and (e) eluting MOMO30 or HEVAR proteinfrom the filter retentate into buffer for storage and/or use.

Identification of MOMO30 Protein Sequence

N-terminal Edman degradation sequencing was carried out to determine theamino terminal sequence of the MOMO30 protein. FIG. 2, panel A showsthat MOMO30 protein comprises the predicted amino acid sequence of SEQID NO. 1. As shown in FIG. 2, panel B, the amino-terminal sequence isvirtually identical to the mature amino-terminal sequence correspondingto several different isolates of a hevamine-A-related protein fromProsopis alba. Hevamines are members of several families of plantchitinases and lysozymes that are important for plant defense againstpathogenic bacteria and fungi and belong to the family 18 glycosylhydrolases. Hevamines are known to hydrolyze linear polysaccharidechains of chitin and peptidoglycan. As described above, the MOMO30protein is heat stable and resistant to most proteases, includingtrypsin, which is used in most liquid chromatography with tandem massspectrometry strategies.

Production of Anti-MOMO30 Antibodies and Detection of MOMO30 Protein

Based on the amino-terminal sequence of the MOMO30 protein, polyclonalantisera was generated in rabbits using a synthetic peptide containingthe amino acid sequence in FIG. 2, panel B sequence. As shown in FIG. 2,panel C, Western blot analysis showed that the anti-MOMO30 antibodydetects a 30 kDa protein from M. balsamina plant extracts, as expected.

MOMO30 Causes Hemagglutination

Co-pending U.S. patent application Ser. No. 16/718,994 characterizesvarious biochemical and functional properties associated with MOMO30,including its ability to bind multiple viruses, including HIV-1, SIV-1and Ebola. As described in the co-pending application, MOMO30 appears tobind sugar groups on viral surface proteins suggesting that MOMO30 hasproperties reminiscent of lectins. Inasmuch as lectins have often beenfound to exhibit hemagglutinin activity, it was of interest toinvestigate whether MOMO30 exhibits hemagglutinin activity too. FIG. 3shows the results of this analysis. In this case, purified MOMO30protein was tested for its ability to agglutinate sheep red blood cells(RBCs). As shown in panel A, a 30 mg/ml stock solution at a dilution of1:512 was found to cause hemagglutination, consistent with lectin-likeactivity.

MOMO30 Stimulates the Activation and Proliferation of T Cells

Inasmuch as lectins are known to function as T cell mitogens, such asphytohemagglutinin A (PHA), it was of interest to examine whether MOMO30can stimulate the activation and proliferation of T cells. Thus, a Tcell activation assay was performed in which a fixed number of Jurkatcells was treated (left to right) with PBS (neg. control, Con), PHA(pos. control), or MOMO30 (FIG. 4). The results of this assay showedthat MOMO30 similarly stimulates the activation and proliferation of Tcells.

MOMO30 Exhibits Mannose-Sensitive Binding to Viral Cell Surface Proteins

Preliminary experiments showed that MOMO30 binds to HIV gp120 (data notshown). To further investigate the nature of the binding between the 30kDa MOMO protein and gp120, MAGI indicator cells were infected with HIVin the presence of the plant extract at increasing concentrations of themonosaccharide mannose. HIV gp120 is known to undergo high-mannoseglycosylation. The results of this analysis in FIG. 5 showed that whilemannose concentrations of 15-20 mM virtually eliminated the ability ofthe MOMO30 protein in the extract to inhibit HIV infection, lowermannose concentrations had little effect on MOMO30's ability to inhibitHIV infection, and higher mannose concentrations had a progressivelydecreased ability to neutralize the inhibitory activity of MOMO30.

To further examine the interaction between MOMO30 and purified gp120,surface plasmon resonance (Biacore) analysis was carried out. Gp120 wasimmobilized on the gold surface of a Biacore chip and increasingconcentrations of MOMO30 protein (from 6.25 nM to 200 nM) were flowedacross the surface and monitored by SPR. After 60 min, regenerationbuffer as added to induce dissociation. The assay was done in triplicateon separate days. The results of this analysis are shown in FIG. 6A andindicate that MOMO30 bound to the surface in a concentration dependentmanner with a KD of 5.8 μM±1.8. To further confirm that binding ofMOMO30 to gp120 is dependent on glycosyl residues, such as mannose, ongp120, a Biocore chip was saturated with gp120 and MOMO30 to formgp120-MOMO30 complexes (FIG. 6B, top curves). The gp120-MOMO30 complexeswere treated with PNGase F to remove sugar residues from gp120 (FIG. 6B,bottom curves). PNGase F is an amidase that works by cleaving betweenthe innermost GlcNAc and asparagine residues of high mannose, hybrid,and complex oligosaccharides from N-linked glycoproteins andglycopeptides, resulting in a deaminated protein or peptide and a freeglycan. In this case, the loss of sugar residues produced a decrease inreflectance units (RU), which reflects a decrease in MOMO30 binding togp120.

Evaluation of Chitinase Activity

Given that the N-terminal amino acid sequence of MOMO30 is consistentwith properties shared by hevamines having chitinase properties, it wasof interest to see whether the MOMO30 protein similarly exhibitschitinase activity. Thus, the Chitinase Microplate Assay Kit(MyBioSource, Inc., San Diego, Calif.) was employed according to themanufacturer's instructions. The results of this analysis confirm thatMOMO30 has chitinase activity (data not shown).

Identification of the HEVAR Gene

To further identify hevamine-related sequences in Momordica balsamina,the following protocol was carried out: (1) isolate total plant RNA fromMomordica balsamina leaves; (2) submit for RNAseq de novo transcriptomeanalysis (GeneWiz); (3) assemble reads in Trinity 2.5 software; (4)search for open reading frames (EMBOSS); (5) translate into proteinsequences (Diamond BLASTx annotation); and (6) search protein sequencesfor hevamine-related sequence motifs.

As shown in FIG. 7, the results of this analysis identified a genecomprising the nucleotide coding sequence of SEQ ID NO: 12, which wastranslated using SnapGene software to reveal a hevamine-related protein(HEVAR) comprising the amino acid coding sequence of SEQ ID NO: 14 (topsequence) shown in FIG. 8.

A nucleic acid database search of the nucleotide coding sequence of SEQID NO: 12 identified a hevamine A-related nucleic acid sequence fromMomordica charantia (NCBI Reference Sequence: XM_022291555.1; SEQ ID NO:16) showing 93% identity to SEQ ID NO: 12. Further, as shown in FIG. 8,an alignment of the amino acid coding sequence of SEQ ID NO: 14 with thetranslation product (SEQ ID NO: 17, via SnapGene) of the Momordicacharantia hevamine A-related nucleotide sequence in FIG. 7 shows 91%identity at the protein level.

As shown in FIG. 8, panel A, the HEVAR amino acid sequence of SEQ ID NO:14 has a signal peptide sequence between amino acid numbers 1-31, whichis removed in the secreted mature protein. FIG. 8, panel B shows theamino acid sequence of the mature HEVAR protein (SEQ ID NO: 15) insecreted form. The nucleotide sequence corresponding to the secretedform of HEVAR is set forth in SEQ ID NO: 13.

FIG. 9 shows an alignment of two conserved regions from the HEVARprotein relative to other hevamine A-related proteins comprising theamino acid sequences set forth in SEQ ID NOs: 18-26.

MOMO30 and HEVAR Binding to SARS-CoV-2 S Protein

The coronavirus (CoV) S protein mediates viral entry into host cells byfirst binding to a host receptor through the receptor-binding domain(RBD) in the 51 subunit and then fusing the viral and host membranesthrough the S2 subunit. Several binding assays may be used to confirmthe ability of MOMO30 and HEVAR to bind coronavirus spike (S) proteins,including SARS-CoV-2 S protein and/or SARS-CoV-2 S1 subunit protein anddetermine the IC50 for MOMO30 and/or HEVAR (i.e., the concentration ofMOMO30 or HEVAR which achieves a half-maximal inhibition). These assaysmay be evaluated for coronavirus binding activity using commerciallyavailable coronavirus reagents, cell lines and/or inhibitor screeningassay kits.

The reagents and kits for these assays may utilize a variety ofSARS-CoV-2 S protein, SARS-CoV-2 S1 subunit (receptor binding domain,RBD) protein-, and ACE2 protein reagents, which may be His-tagged,Fc-tagged, Avi-tagged, or biotin-labeled in order to facilitatedetection of binding on microtiter plates and the like using suitablecolorimetric or chemoluminescent substrates (BPS Bioscience, San Diego,Calif.).

In one embodiment, ACE2 protein is coated onto a 96-well microtiterplate and then incubated with a composition containing an aqueousMOMO30-containing plant extract, a HEVAR-containing plant extract,MOMO30 protein or HEVAR protein pre-incubated with His-, His-Avi- orFc-tagged CoV-2 Spike (S) protein (or tagged versions of the 51 subunitprotein), followed by recovery and detection of bound complexes usingsuitable detection reagents known in the art. The Avi-tag further allowsfor biotinylation of the CoV-2 fusion protein, which can facilitatebinding to e.g., streptavidin-HRP conjugates for detection of binding.

Alternatively, His-, His-Avi- or Fc-tagged S protein or S1 protein iscoated onto a 96-well microtiter plate and then incubated with acomposition containing an aqueous MOMO30-containing plant extract, aHEVAR-containing plant extract, MOMO30 protein or HEVAR proteinpre-incubated with His-, His-Avi- or Fc-tagged ACE2 protein, followed byrecovery and detection of bound complexes using suitable detectionreagents and conjugates known in the art.

In one embodiment, the MOMO30-containing plant extract, aHEVAR-containing plant extract, MOMO30 protein or HEVAR protein isincubated with purified CoV-2 spike (S) protein or purified CoV-2receptor binding domain (RBD) and loaded on a non-denaturingpolyacrylamide gel. The production of a band-shift compared to controlsis consistent with binding of the MOMO30 or HEVAR protein to the Sprotein or S1 subunit.

In another embodiment, the MOMO30-containing plant extract, aHEVAR-containing plant extract, MOMO30 protein or HEVAR protein isevaluated for its ability to inhibit binding of purified fluorescentlylabeled CoV-2 S protein or CoV-2 S1 (RBD) subunit to its co-receptorACE2 in ACE2-expressing cells. In this assay, purified fluorescentlylabeled CoV-2 S protein or CoV-2 S1 subunit is added to ACE2-expressingcells in the presence of increasing amounts of MOMO30 or HEVAR proteinor a negative control (PBS only). Specific binding is shown bydemonstrating that increasing concentrations MOMO30 or HEVAR proteinlead to progressively less attachment of the fluorescently labeledCoV-2-S protein or CoV-2-S1 subunit to the ACE2 expressing cells.

In another embodiment, the interaction between MOMO30 or HEVAR proteinand purified coronavirus S1 protein is evaluated by surface plasmonresonance (Biacore). In this assay, CoV-2-S protein or S1 protein isimmobilized on the gold surface of a Biacore chip and increasingconcentrations of MOMO30 or HEVAR protein (from e.g., 6.25 nM to 200 nM)are flowed across the surface and monitored by SPR. After 60 min,regeneration buffer is added to induce dissociation.

To confirm that MOMO30 or HEVAR protein binds to high mannose residuesin CoV-2-S protein (or CoV-2-S1 subunit), a Biocore chip is saturatedwith CoV-2-S protein (or CoV-2-S1 subunit) and MOMO30 or HEVAR to formCoV-2-S protein-MOMO30- or HEVAR complexes. In addition, the CoV-2-Sprotein-MOMO30/HEVAR complexes can be treated with PNGase F to removesugar residues from CoV-2-S protein (or CoV-2-S1 subunit). PNGase F isan amidase that works by cleaving between the innermost GlcNAc andasparagine residues of high mannose, hybrid, and complexoligosaccharides from N-linked glycoproteins and glycopeptides,resulting in a deaminated protein or peptide and a free glycan. In thiscase, the loss of sugar residues produces a decrease in reflectanceunits (RU), which reflects a decrease in MOMO30 or HEVAR binding toCoV-2-S protein. Reagents and cell lines for carrying out the aboveexperiments may be obtained from BPS Bioscience (San Diego, Calif.) andCreative Biogene (Shirley, N.Y.).

Functional Activity of MOMO30- or HEVAR Containing Cell Extracts andProteins

MOMO30- or HEVAR-containing cell extracts and/or proteins are tested foranti-SARS-CoV-2 activity using a functional assay evaluating the abilityof MOMO30 or HEVAR to inhibit CoV-2 replication. The functional assaysdescribed below further allow for the generation of a dose-responsecurve reflecting the degree of CoV-2 inhibition, including an 1050determination for MOMO30 or HEVAR.

In one embodiment, a MOMO30-containing plant extract, HEVAR-containingplant extract, purified MOMO30 protein or purified HEVAR protein istested for inhibitory activity by an in vitro plaque reduction assayusing SARS-CoV-2 infected Vero E6 cells, a monkey kidney cell line,which is known to express the ACE2 receptor. Briefly, Vero E6 cells areplated onto 12-well tissue culture plates and incubated overnight toallow for adherence to the plates. Serial dilutions of MOMO30 or HEVARin cell maintenance media are then incubated with a defined amount ofSARS-CoV-2 for one hour in the absence of Vero E6 cells. Negativecontrol solutions include SARS-CoV-2 incubated for 1 hr in cellmaintenance media without MOMO30, HEVAR or cells. Following the one hourincubation, the cell maintenance media is removed from the Vero E6seeded plates and replaced with the pre-incubated solutions ofMOMO30/SARS-CoV-2/cell media (test), HEVAR/SARS-CoV-2/cell media (test)and/or SARS-CoV-2/cell media (negative control). The cells are thenincubated for 1 hr to allow adsorption of virus to the cells. Followingthe 1 hr incubation, the suspension is removed and methylcelluloseoverlays containing matched concentrations of MOMO30 or HEVAR are addedto each well. The plates are incubated for 3 days, inactivated and thenstained with crystal violet stain. Dose response curves are thengenerated based on the degree of replication inhibition in each wellcompared to the corresponding negative controls (i.e., absence of MOMO30or HEVAR).

In another embodiment, a MOMO30- or HEVAR-containing plant extract orpurified MOMO30 protein is tested for inhibitory activity usinglentivirus-based, VSV-based or MuLV-based virus particles operablylinked to a luciferase reporter that are pseudotyped with a CoV Spike(S) protein, such as SARS-CoV-2 S protein. More particularly, the assayevaluates the ability of MOMO30 or HEVAR to block expression of theluciferase reporter in ACE2-expressing cells infected with theS/S1-pseudotyped lentivirus reporter. A “bald” or non-pseudotypedcontrol containing the luciferase reporter alone can be used as anegative control.

The ACE2-expressing cells or cell lines are infected with thepseudotyped or non-pseudotyped virus particles in the presence ofincreasing concentrations of MOMO30 or HEVAR. When using cellsexhibiting low or no ACE2 expression, the pseudotyped and/ornon-pseudotyped virus particles are co-infected with an expressionconstruct, such replication-defective HIV-1 particles engineered toexpress human ACE2. A lentivirus-based luciferase reporter system forcarrying out this assay includes pseudotyped (CoV-2 S protein)lentivirus reporters, non-pseudotyped lentivirus reporters (negativecontrol), and ACE2-expressing lentiviruses (BPS Bioscience, San Diego,Calif., BPS #s 79942, 79943 and 79944). Additional reagents and celllines for carrying out the above experiments may be obtained from BPSBioscience (San Diego, Calif.) and Creative Biogene (Shirley, N.Y.).

To further confirm the binding of MOMO30 or HEVAR to high mannoseresidues in coronavirus S proteins, the above described functional assaymay be carried out at increasing concentrations of the monosaccharidemannose. It is predicted that increasing mannose concentrations willprogressively eliminate the ability of the MOMO30 or HEVAR protein toinhibit CoV-2 replication in Vero E6 cells and inhibit luciferase orβ-gal expression from the reporter.

Efficacy Study of SARS CoV-2 Patients Treated with a MOMO30 or HEVARHerbal Tea

In one embodiment, to evaluate of therapeutic efficacy of the MOMO30 orHEVAR protein, SARS CoV-2-infected patients are orally administered anherbal tea containing MOMO30 or HEVAR for a period of 6 months duringwhich no other antiviral agents are administered. During this 6 monthtreatment period, the patients' viral loads and CD4+ lymphocyte countsare monitored. The results of this study are expected to show asignificant reduction in average viral load accompanied by increasedCD4+ cell counts increased over this same period.

In certain embodiments, to evaluate the therapeutic efficacy of theMOMO30 or HEVAR protein and facilitate a determination of effectivedosages and administration protocols for the pharmaceutical compositionsof the present application, an in vivo SARS-CoV-2 Syrian hamster modelcan be employed prior to human clinical trials. Use of this model canaddress key aspects of SARS-CoV-2 pathology following delivery of theprotein or nucleic acid-based compositions of the present application,such as evaluation of viral loads and alleviation of the occurrence andseverity of downstream effects, including the cytokine storm associatedwith SARS-CoV-2 infections. Syrian hamsters are permissive to SARS-CoV-2and develop mild lung disease similar to the disease observed inearly-stage COVID-19 patients.

Briefly, 6-8 week old female Syrian hamsters are anesthetized withketamine/xylazine/atropine and inoculated intranasally (twice daily)with 50 μL containing about 1×10⁵ TCID₅₀ (median tissue cultureinfectious dose) of a SARS-CoV-2 strain (day 0). Beginning 2 h beforeinfection, animals are administered twice daily with a pharmaceuticalcomposition described herein (positive control) and/or thepharmaceutically acceptable carrier(s) used in the pharmaceuticalformulation (negative control). The pharmaceutical composition may bedelivered orally, intranasally, by intratracheal instillation or byaerosol inhalation. Hamsters are monitored for appearance, behavior andweight. At day 2 post infection (pi) (day 3), 6 h following the 5thdose, hamsters are euthanized by IP injection of 500 μL Dolethal (200mg/mL sodium pentobarbital, Vétoquinol SA). Lungs are collected andviral RNA and infectious virus are quantified by RT-qPCR and end-pointvirus titration, respectively. In addition, blood samples and lungtissue samples are collected and evaluated for cytokine levels andpharmacokinetic analysis of the HEVAR or MOMO30 protein of the presentapplication. The results of these studies are expected to show at leasta 2 or 3 log₁₀ reduction in viral RNA copies per mg of lung tissuecompared to the vehicle. Additionally, the results are expected to showa reduction of at least 10%, 20%, 50% or 80% in cytokine levels for oneor more of IL-6, IL-1β, IL-2, IL-10, IFN-γ, TNF-α, and GM-CSF.

While various embodiments have been described above, it should beunderstood that such disclosures have been presented by way of exampleonly and are not limiting. Thus, the breadth and scope of the subjectcompositions and methods should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the components and steps in any sequence which iseffective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1. A method for treating or preventing a Severe Acute RespiratorySyndrome Corona Virus-2 (SARS-CoV-2) infection, comprising orallyadministering to a subject in need thereof a composition comprising: aneffective amount hevamine related (HEVAR) protein which comprises anamino acid sequence that is at least 90% identical to SEQ ID NO: 15; andat least one pharmaceutically acceptable carrier.
 2. The method of claim1, wherein the amino acid sequence is 95%, 99% or 100% identical to SEQID NO:
 15. 3. The method of claim 1, further comprising a secondantiviral agent.
 4. The method of claim 1, wherein the composition is ina dried form.
 5. The method of claim 4, wherein the dried form comprisesa capsule or tablet.
 6. The method of claim 1, wherein the compositionis in a liquid form.
 7. The method of claim 1, wherein the compositioncomprises an herbal tea.
 8. The method of claim 1, wherein the HEVARprotein is prepared by the steps of: (a) drying a plant comprising HEVARprotein; (b) extracting the dried plant in aqueous media; (c) lysingcells from the extracted plant to form a plant cell lysate; and (d)centrifuging the plant cell lysate to remove debris and particulates toform a clarified plant cell lysate; and (e1) passing the plant celllysate through a molecular weight cut-off filter and collecting theHEVAR-containing retentate, or (e2) purifying the HEVAR protein from theclarified plant cell lysate by immunoaffinity purification using ananti-HEVAR antibody.
 9. A method for reducing or preventing the severityof the cytokine storm in a SARS-CoV-2 infected patient comprising:administering an effective amount of a pharmaceutical composition apharmaceutical composition comprising an effective amount of a hevamineA-related plant protein which comprises an amino acid sequence that hasat least 90% identical to SEQ ID NO: 15; and at least onepharmaceutically acceptable carrier, wherein the method results in atleast a 20% reduction in cytokine levels for one or more of IL-6, IL-1β,IL-2, IL-10, IFN-γ, TNF-α, GM-CSF, or VEGF.
 10. A pharmaceuticalcomposition comprising: a substantially pure HEVAR protein whichcomprises an amino acid sequence at least 95% or 99% identical to SEQ IDNO: 15; and at least one pharmaceutically acceptable carrier.
 11. Thecomposition of claim 10, wherein the HEVAR protein comprises the aminoacid sequence of SEQ ID NO:
 15. 12. The composition of claim 10, furthercomprising a second antiviral agent.
 13. The composition of claim 10,wherein the composition comprises a capsule or tablet.
 14. Thecomposition of claim 10, wherein the composition is in a liquid form.15. The composition of claim 10, wherein the composition comprises anherbal tea.
 16. A method for treating or preventing a SARS-CoV-2infection, comprising administering to a subject in need thereof apharmaceutical composition comprising: an effective amount of anexpression vector operatively linked to a nucleic acid having anucleotide sequence that is at least 95% identity to SEQ ID NO: 12 orSEQ ID NO: 13; and at least one pharmaceutically acceptable carrier. 17.The method of claim 16, wherein the nucleic acid comprises thenucleotide sequence of SEQ ID NO:
 13. 18. The method of claim 16,wherein the expression vector is an adeno-associated virus vector. 19.The method of claim 16, wherein the pharmaceutical composition comprisesa cell transformed with expression vector.